Alkyl, azido, alkoxy, and fluoro-substituted and fused quinoxalinediones and the use thereof as glycine receptor antagonist

ABSTRACT

Methods of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia, and surgery, as well as treating neurodegenerative diseases including Alzheimer&#39;s disease, amyotrophic lateral sclerosis, Huntington&#39;s disease, and Down&#39;s syndrome, treating or preventing the adverse consequences of the hyperactivity of the excitatory amino acids, as well as treating anxiety, chronic pain, convulsions, and inducing anesthesia are disclosed by administering to an animal in need of such treatment an alkyl or azido-substituted 1,4-dihydroquinoxaline-2,3-dione or pharmaceutically acceptable salts thereof, which have high binding to the glycine receptor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/376,536, filed Aug. 18, 1999, U.S. Pat. No. 6,147,075, which is adivisional of U.S. application Ser. No. 08/792,872, filed Jan. 31, 1997,now U.S. Pat. No. 5,977,107, which is a divisional of U.S. applicationSer. No. 08/289,603, filed Aug. 11, 1994, now U.S. Pat. No. 5,631,373,which is a continuation-in-part of U.S. application Ser. No. 08/208,878,filed Mar. 11, 1994, now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 08/148,268, filed Nov. 5, 1993, now abandoned,and a continuation-in-part of U.S. application Ser. No. 08/148,259,filed Nov. 5, 1993, now U.S. Pat. No. 5,514,680.

This invention was made with government support under grant numbers NIDADA 06727, NIDA DA 06356, and NIDA DA 06726 awarded by the NationalInstitutes of Health. The U.S. government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the field of medicinal chemistry and relatesto compounds that have a high affinity for the glycine binding site,lack PCP side effects, and cross the blood brain barrier at high levels.In particular, the present invention relates to novel alkyl, azido,alkoxy, fluoro-substituted, and fused 1,4-dihydroquinoxaline-2,3-dionesand their use to treat or prevent neuronal degeneration associated withischemia, pathophysiologic conditions associated with neuronaldegeneration, convulsions, anxiety, chronic pain, and to induceanesthesia.

2. Description of the Related Art

Glutamate is thought to be the major excitatory neurotransmitter in thebrain. There are three major subtypes of glutamate receptors in the CNS.

These are commonly referred to as kainate, AMPA, andN-methyl-D-aspartate (NMDA) receptors (Watkins and Olverman, Trends inNeurosci. 7:265-272 (1987)). NMDA receptors are found in the membranesof virtually every neuron in the brain. NMDA receptors are ligand-gatedcation channels that allow Na⁺, K⁺, and Ca⁺⁺ to permeate when they areactivated by glutamate or aspartate (non-selective, endogenous agonists)or by NMDA (a selective, synthetic agonist) (Wong and Kemp, Ann. Rev.Pharmacol. Toxicol. 31:401-425 (1991)).

Glutamate alone cannot activate the NMDA receptor. In order to becomeactivated by glutamate, the NMDA receptor channel must first bindglycine at a specific, high affinity, glycine binding site that isseparate from the glutamate/NMDA binding site on the receptor protein(Johnson and Ascher, Nature 325:329-331 (1987)). Glycine is therefore anobligatory co-agonist at the NMDA receptor/channel complex (Kemp, J. A.,et al., Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)).

In addition to the binding sites for glutamate/NMDA and glycine, theNMDA receptor carries a number of other functionally important bindingsites. These include binding sites for Mg⁺⁺, Zn⁺⁺, polyamines,arachidonic acid, and phencyclidine (PCP) (Reynolds and Miller, Adv. inPharmacol. 21:101-126 (1990); Miller, B., et al., Nature 355:722-725(1992)). The PCP binding site—now commonly referred to as the PCPreceptor—is located inside the pore of the ionophore of the NMDAreceptor/channel complex (Wong, E. H. F., et al., Proc. Natl. Acad. Sci.USA 83:7104-7108 (1986); Huettner and Bean, Proc. Natl. Acad. Sci. USA85:1307-1311 (1988); MacDonald, J. F., et al., Neurophysiol. 58:251-266(1987)). In order for PCP to gain access to the PCP receptor, thechannel must first be opened by glutamate and glycine. In the absence ofglutamate and glycine, PCP cannot bind to the PCP receptor although somestudies have suggested that a small amount of PCP binding can occur evenin the absence of glutamate and glycine (Sircar and Zukin, Brain Res.556:280-284 (1991)). Once PCP binds to the PCP receptor, it blocks ionflux through the open channel. Therefore, PCP is an open channel blockerand a non-competitive glutamate antagonist at the NMDA receptor/channelcomplex.

One of the most potent and selective drugs that bind to the PCP receptoris the anticonvulant drug MK801. This drug has a K_(d) of approximately3 nM at the PCP receptor (Wong, E. H. F., et al., Proc. Natl. Acad. Sci.USA 83:7104-7108 (1986)).

Both PCP and MK801 as well as other PCP receptor ligands, e.g., Ifdextromethorphan, ketamine, and N,N′-disubstituted guanidines, haveneuroprotective efficacy both in vitro and in vivo (Gill, R., et al., J.Neurosci. 7:3343-3349 (1987); Keana, J. F. W., et al., Proc. Natl. Acad.Sci. USA 86:5631-5635 (1989); Steinberg, G. K., et al., NeuroscienceLett. 89: 193-197 (1988); Church, J., et al., In: Sigma andPhencyclidine-Like Compounds as Molecular Probes in Biology, Domino andKamenka, eds., Ann Arbor: NPP Books, pp. 747-756 (1988)). Thewell-characterized neuroprotective efficacy of these drugs is largelydue to their capacity to block excessive Ca⁺⁺ influx into neuronsthrough NMDA receptor channels, which become over activated by excessiveglutamate release in conditions of brain ischemia (e.g. in stroke,cardiac arrest ischemia etc.) (Collins, R. C., Metabol. Br. Dis.1:231-240 (1986); Collins, R. C., et al., Annals Int. Med. 110:992-1000(1989)).

However, the therapeutic potential of these PCP receptor drugs asischemia rescue agents in stroke has been severely hampered by the factthat these drugs have strong PCP-like behavioral side effects(psychotomimetic behavioral effects) which appear to be due to theinteraction of these drugs with the PCP receptor (Tricklebank, M. D., etal., Eur. J. Pharmacol. 167:127-135 (1989); Koek, W., et al., J.Pharmacol. Exp. Ther. 245:969 (1989); Willets and Balster,Neuropharmacology 27:1249 (1988)). These PCP-like behavioral sideeffects appear to have caused the withdrawal of MK801 from clinicaldevelopment as an ischemia rescue agent. Furthermore, these PCP receptorligands appear to have considerable abuse potential as demonstrated bythe abuse liability of PCP itself.

The PCP-like behavioral effects of the PCP receptor ligands can bedemonstrated in animal models: PCP and related PCP receptor ligandscause a behavioral excitation (hyperlocomotion) in rodents (Tricklebank,M. D., et al., Eur. J. Pharmacol. 167:127-135 (1989)) and acharacteristic catalepsy in pigeons (Koek, W., et al., J. Pharmacol.Exp. Ther. 245:969 (1989); Willets and Balster, Neuropharmacology27:1249 (1988)); in drug discrimination paradigms, there is a strongcorrelation between the PCP receptor affinity of these drugs and theirpotency to induce a PCP-appropriate response behavior (Zukin, S. R., etal., Brain Res. 294:174 (1984); Brady, K. T., et al., Science 215:178(1982); Tricklebank, M. D., et al., Eur. J. Pharmacol. 141:497 (1987)).

Drugs acting as competitive antagonists at the glutamate binding site ofthe NMDA receptor, such as, CGS 19755 and LY274614, also haveneuroprotective efficacy because these drugs—like the PCP receptorligands—can prevent excessive Ca⁺⁺ flux through NMDA receptor/channelsin ischemia (Boast, C. A., et al., Brain Res. 442:345-348 (1988);Schoepp, D. D., et al., J. Neural. Trans. 85:131-143 (1991)). However,competitive NMDA receptor antagonists also have PCP-like behavioralside-effects in animal models (behavioral excitation, activity in PCPdrug discrimination tests) although not as potently as MK801 and PCP(Tricklebank, M. D., et al., Eur. J. Pharmacol. 167:127-135 (1989)).

An alternate way of inhibiting NMDA receptor channel activation is byusing antagonists at the glycine binding site of the NMDA receptor.Since glycine must bind to the glycine site in order for glutamate toeffect channel opening (Johnson and Ascher, Nature 325:329-331 (1987);Kemp, J. A., et al., Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)), aglycine antagonist can completely prevent ion flux through the NMDAreceptor channel—even in the presence of a large amount of glutamate.

Recent in vivo microdialysis studies have demonstrated that, in the ratfocal ischemia model, there is a large increase in glutamate release inthe ischemic brain region with no significant increase in glycinerelease (Globus, M. Y. T., et at., J. Neurochemn. 57:470-478 (1991)).Thus, theoretically, glycine antagonists should be very powerfulneuroprotective agents because they can prevent the opening of NMDAchannels by glutamate non-competitively and, therefore, unlikecompetitive NMDA antagonists, do not have to overcome the largeconcentrations of endogenous glutamate that are released in the ischemicbrain region.

Furthermore, because glycine antagonists act at neither theglutamate/NMDA nor the PCP binding sites to prevent NMDA channelopening, these drugs might not cause the PCP-like behavioral side effectseen with both PCP receptor ligands and competitive NMDA receptorantagonists (Tricklebank, M. D., et al., Eur. J. Pharmcol. 167:127-135(1989); Koek, W., et al., J. Pharmacol. Exp. Ther. 245:969 (1989);Willets and Balster, Neuropharmacology 27:1249 (1988); Tricklebank, M.D., et al., Eur. J. Pharmacol. 167:127-135 (1989); Zukin, S. R., et al.,Brain Res. 294:174 (1984); Brady, K. T., et al., Science 215:178 (1982);Tricklebank, M. D., et al., Eur. J. Pharmacol. 141:497 (1987)). Thatglycine antagonists may indeed be devoid of PCP-like behavioral sideeffects has been suggested by recent studies in which available glycineantagonists were injected directly into the brains of rodents withoutresulting in PCP-like behaviors (Tricklebank, M. D., et al., Eur. J.Pharmacol. 167:127-135 (1989)).

However, there have been two major problems that have prevented thedevelopment of glycine antagonists as clinically useful neuroprotectiveagents:

A. Most available glycine antagonists with relatively high receptorbinding affinity in vitro such as 7-Cl-kynurenic acid (Kemp, J. A., etal., Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)),5,7-dichlorokynurenic acid (McNamara, D., et al., Neuroscience Lett.120:17-20 (1990)) and indole-2-carboxylic acid (Gray, N. M., et al., J.Med. Chem. 34:1283-1292 (1991)) cannot penetrate the blood/brain barrierand therefore have no utility as therapeutic agents;

B. The only available glycine antagonist that sufficiently penetratesthe blood/brain barrier—the drug HA-966 (Fletcher and Lodge, Eur. J.Pharmacol. 151:161-162 (1988))—is a partial agonist with only micromolaraffinity for the glycine binding site. A neuroprotective efficacy forHA-966 in vivo has, therefore, not been demonstrated, nor has it beendemonstrated for the other available glycine antagonists because theylack bioavailability in vivo.

However, one recent success in identifying orally active glycinereceptor antagonists was reported by Kulagowski et al., J. Med. Chem.37:1402-1405 (1994), who disclose that 3-substituted4-hydroxyquinoline-2(1H)-diones are selective antagonists possessingpotent potent in vivo activity.

There have been a number of reports in the literature of substituted1,4-dihydroquinoxaline-2,3-diones that are useful for treatingpathophysiologic conditions mediated by the non-NMDA, NMDA, and glycinereceptors. For example, U.S. Pat. No. 4,975,430 discloses1,4-dihydroquinoxaline-2,3-dione compounds of the formula:

wherein each X is independently nitro or cyano and wherein each Y isindependently H, lower alkyl, lower alkoxy, or CF₃. These compounds arereportedly useful for the treatment of neuronal conditions associatedwith stimulation of the NMDA receptor.

U.S. Pat. No. 3,962,440 discloses 1,4-dihydroquinoxaline-2,3-dionecompounds having the formula:

wherein, R¹ can be hydrogen or methyl, R_(n) can be lower alkyl loweralkoxy, lower alkylthio, cyclopropyl, nitro, cyano, halogen, fluoroalkylof C₁-C₂ (trifluoromethyl) amino, or substituted amino, and n can be 0,1, or 2. These compounds are reportedly useful as hypnotic agents. itU.S. Pat. No. 4,812,458 discloses 1,4-dihydroquinoxaline-2,3-dionecompounds having the formula:

wherein R¹ is halogen, cyano, trifluoromethyl, ethynyl, or N₃, and ² isSO₂C₁₋₃-alkyl, trifluoromethyl, nitro, ethynyl, or cyano. Thesecompounds are reportedly useful for treatment of indications caused byhyperactivity of the excitatory neurotransmitters, particularly thequisqualate receptors, and as neuroleptics.

U.S. Pat. No. 4,659,713 discloses 1,4-dihydroquinoxaline-2,3-dionecompounds having the formula:

wherein X represents hydrogen, chloro, bromo, fluoro, iodo,trichloromethyl, dichlorofluoromethyl, difluoromethyl, ortrifluoromethyl, and n represents I or 2. These compounds are reportedlyuseful for the control of coccidiosis in animals.

U.S. Pat. No. 4,948,794 discloses 1,4-dihydroquinoxaline-2,3-dionecompounds having the formula:

wherein

R¹ is C₁₋₁₂-alkyl, which may, optionally, be substituted by hydroxy,formyl, carboxy, carboxylic esters, amides, or amines, C₃₋₈-cycloalkyl,aryl, aralkyl; and wherein R⁶ is, hydrogen, halogen, CN, CF₃, NO₂, orOR′, wherein R′ is C₁₋₄-alkyl, and R⁵, R⁷, and R⁸ are hydrogen, providedR⁶ is not CF₃, OCH₃, NO₂, Cl, or Br when R¹ is CH₃; or

R⁶ and R⁷ independently are NO₂, halogen, CN, CF₃, or OR′, wherein R′ isC₁₋₄-alkyl, and R⁵ and R⁸ are each hydrogen; or

R⁵ and R⁶ together form a further fused aromatic ring, which may besubstituted with halogen, NO₂, CN, CF₃, or OR′, wherein R′ isC₁₋₄-alkyl; or

R⁷ and R⁸ together form a further fused aromatic ring, which may besubstituted with halogen, NO₂, CN; CF₃, or OR′, wherein R′ isC₁₋₄-alkyl, and R⁵ and R⁶ independently are hydrogen, halogen, CN, CF₃,NO₂, or OR′, wherein R′ is C₁₋₄-alkyl. These compounds are reportedlyuseful for the treatment of indications caused by hyperactivity of theexcitatory neurotransmitters, particularly the quisqualate receptors,and as neuroleptics.

Yoneda and Ogita, Biochem. Biophys. Res. Commun. 164:841-849 (1989),disclose that the following 1,4-dihydroquinoxaline-1,2-dionecompetitively displaced the strychnine-insensitive binding of[³H]glycine, without affecting the other binding sites on the NMDAreceptor complex:

According to the authors, the structure-activity relationships amongquinoxalines clearly indicates that both chloride groups of thepositions 6 and 7 in the benzene ring are crucial for the antagonistpotency against the Gly sites. Removal of one chloride from the moleculeresults in a 10-fold reduction in the affinity for Gly sites.

Kleckner and Dingledine, Mol. Pharm. 36:430-436 (1989), disclose that6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione and6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione are more potentantagonists of kainate than glycine, but substitution of Cl at the6-position and especially at the 6- and 7-positions increases potency atthe glycine site. In addition, the authors suggest that antagonists ofthe glycine site might be effective against NMDA receptor-mediatedneuropathologies.

Rao, T. S. et at., Neuropharmacology 29:1031-1035 (1990), disclose that6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione and7-cyano-6-nitro-1,4-dihydroquinoxaline-2,3-dione antagonize responsesmediated by NMDA-associated glycine recognition sites in vivo.

Pellegrini-Giampietro, D. E. et al., Br. J. Pharmacol. 98:1281-1286(1989), disclose that 6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dioneand 6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione can antagonize theresponses to L-glutamate by interacting with the glycine recognitionsites of the NMDA receptor ion channel complex.

Ogita and Yoneda, J. Neurochem. 54:699-702 (1990), disclose that6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione is a competitiveantagonist specific to the strychnine-insensitive [³H] glycine bindingsites on the NMDA receptor complex. According to the authors, the twochloride radicals at the 6- and 7-positions in the benzene ring of thequinoxaline are crucial for the antagonistic potency against the glycinebinding sites.

Kessler, M. et al., Brain Res. 489:377-382 (1989), disclose that6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione and6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione inhibit [³H] glycinebinding to the strychnine-insensitive glycine binding sites associatedwith NMDA receptors.

European Patent Application Publication No. 0 377 112, published Jul.11, 1990, discloses 1,4-dihydroquinoxaline-2,3-dione compounds havingthe formula:

wherein, inter alia, R¹ can be hydroxy, alkoxy, aryloxy, aralkyloxy,cycloalkylalkoxy, cycloalkoxy, or alkanoyloxy; and R⁵, R⁶, R⁷ and R⁸ canbe independently hydrogen, nitro, halogen, cyano, trifluoromethyl,SO₂NR′R′, SO₂R′ or OR′, wherein R′ is hydrogen or C₁₋₄ alkyl. Thesecompounds are reportedly useful for the treatment of indications causedby hyperactivity of the excitatory neurotransmitters, particularly thequisqualate receptors, and as neuroleptics.

Lester, R. A. et al., Mol. Pharm. 35:565-570 (1989), disclose that6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione antagonizes NMDAreceptor-mediated responses by a competitive interaction of the glycinebinding site.

Patel, J. et al., J. Neurochem. 55:114-121 (1990), disclose that theneuroprotective activity of 6,7-dinitro-1,4-dihydroquinoxaline-2,3-dioneis due to antagonism of the coagonist activity of glycine at the NMDAreceptor-channel complex.

Horner, L. et al., Chem. Abstracts 48:2692 (1953) disclose 6,8-dinitro1,4-dihydroquinoxaline-2,3-dione.

Cheeseman, G. W. H., J. Chem Soc.:1170-1176 (1962), discloses6,7-dibromo-2,3-dihydroxyquinoxaline (also known as6,7-dibromo-1,4-dihydroquinoxaline-2,3-dione).

Honore, T. et al., Science 241.701-703 (1988), disclose that6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione and7-cyano-6-nitro-1,4-dihydroquinoxaline-2,3-dione are potent non-NMDAglutamate receptor antagonists.

Sheardown, M. J. et al., Eur. J. Pharmacol. 174:197-204 (1989), disclosethat 5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione is a potent-antagonistof the strychnine insensitive glycine receptor and has anticonvulsantproperties. However, Sheardown et al. also disclose that5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione as well as DNQX and CNQXhave poor access to the central nervous system.

International Application Publication No. WO91/13878 discloses thefollowing N-substituted 1,4-dihydroquinoxaline-2,3-diones, which bind tothe glycine receptor:

wherein R represents hydrogen, C₁₋₆ alkyl, or aralkyl, and n is aninteger from 0 to 5; R⁴ represents hydrogen or hydroxy; R⁵, R⁶, R⁷, andR⁸ independently represent hydrogen, nitro, halogen, alkoxy, aryloxy,aralkoxy, C₁₋₆-alkyl, or aryl; R⁹ represents hydrogen, lower alkyl, oraryl; R¹⁰ represents hydrogen, or alkyl, and pharmaceutically acceptablesalts thereof.

Leeson et al., J. Med. Cam 34:1243-1252 1991), disclose a number ofderivatives of the nonselective excitatory amino acid antagonistkynurenic acid. Also disclosed are a number of structurally relatedquinoxaline-2,3-diones that are also glycine/NMDA antagonists, but arenot selective and are far less potent than the kynurenic acidderivatives. The quinoxaline-2,3-diones have the structure:

wherein R is H, 5-Cl, 7-Cl, 5,7-Cl₂,6,7-Cl₂,6,7-(CH₃)₂,6-NO₂, or6,7-(NO₂)₂. Also disclosed are a number of N-methyl derivatives.

Epperson et al., Bioorganic & Medicinal Chemistry Letters,3(12):2801-2804 (1993) report the synthesis and amino acid pharmacologyof twelve N-substituted quinoxalinediones. In particular, compounds ofthe structure

are reported to have significant antagonism at both the AMPA andglycine-site NMDA receptors. The functional antagonism of 4a has beendemonstrated. By way of background, the authors teach thatquinoxalinediones such as 6,7-dinitroquinoxaline-2,3-dione and6-cyano-7-nitroquinoxaline-2,3-dione and6-cyano-7-dinitroquinoxaline-2,3-dione have been shown to be AMPA(Honore et al., Science 241:701 (1988)) as well as glycine antagonists(Birch et al., Eur. J. Pharmacol. 156:177 (1988)), and also to beneuroprotective in vitro grandson, et al., J. Neurochem. 53:297 (1989))and the AMPA selective quinoxalinedione2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline has been shownto be neuroprotective in cerebral ischemia models (Sheardown et al.,Science 247:571 (1990)).

For a recent review on glycine antagonists, reference is made to Leeson,P. D., “GlycineSite N-Methyl-D-Aspartate Receptor Antagonists,” Chapter13 in Drug Design for Neurascience, Kozikowski, A. P. (ed.), RavenPress, New York, pp. 338-381 (1993).

A need continues to exist for potent and selective glycine/NMDAantagonists that:

lack the PCP-like behavioral side effects common to the PCP-like NMDAchannel blockers, such as, MK801, or to the competitive NMDA receptorantagonists, such as, CGS19755;

show potent anti-ischemic efficacy because of the non-competitive natureof their glutamate antagonism at the NMDA receptor;

cross the blood-brain barrier at levels sufficient for efficacy;

have utility as novel anticonvulsants with fewer side-effects than thePCP-like NMDA channel blockers or the competitive NMDA antagonists;

help in defining the functional significance of the glycine binding siteof the NMDA receptor in vivo.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a class of compoundsthat exhibit high affinity for the strychnine-insensitive glycinebinding site, that do not exhibit PCP side effects, and that cross theblood brain barrier at high levels. Such compounds include alkyl, azido,alkoxy, hydroxy, and fluoro-substituted 1,4-quinoxaline-2,3-diones, aswell as ring fused 1,4-quinoxaline-2,3-diones. The high affinity ofalkyl-substituted 1,4-quinoxaline-2,3-diones to the glycine binding sitewas unexpected, in view of the many reports in the literature that thepresence of electron withdrawing groups on the benzene nucleus isrequired for high binding affinity and that alkyl and alkoxy groups areelectron donating and azido is only weakly electron withdrawing.

The invention also relates to a method of treating or preventingneuronal loss associated with stroke, ischemia, CNS trauma,hypoglycemia, and surgery; treating neurodegenerative diseases,including, Alzheimer's disease, amyotrophic lateral sclerosis,Huntington's disease, and Down's syndrome; treating or preventing theadverse consequences of the overstimulation of the excitatory aminoacids; treating anxiety, convulsions, chronic pain, or psychosis;preventing opiate tolerance; or inducing a hypnotic effect or anesthesiacomprising administering to an animal in need of such treatment orprevention a substituted 1,4-dihydroquinoxaline-2,3-dione, as definedherein, having high affinity for the glycine binding site and thecapability of crossing the blood brain barrier at high levels, whilelacking PCP side effects.

The compounds of the present invention include those having the FormulaI:

or a tautomer thereof;

wherein

R is hydrogen, hydroxy, amino, —CH₂CONHAr, —NHCONHAr, —NHCOCH₂Ar,—COCH₂Ar, wherein Ar is an aryl group, or a radical having the formula:

wherein R⁶ is hydrogen, lower alkyl of 1-6 carbon atoms or aryl; R⁷ ishydrogen or lower alkyl of 1-6 carbon atoms; n is an integer from 0 to5; and R⁸ is hydrogen, C₁₋₆ alkyl, or aralkyl;

R¹, R², R³, and R⁴ are independently selected from the group consistingof hydrogen, hydroxy, acyloxy, aralkoxy, amino, alkanoylamino, halo,haloalkyl, nitro, alkyl, alkoxy, carboxy, alkanoyl, thioalkyl,alkoxyalkyl, aryloxyalkyl, arylalkyl, alkenyl, alkynyl, arylalkenyl,arylalkynyl, cyano, cyanomethyl, dicyanomethyl, cyanoamino,dicyanoamino, or azido; or where R¹ and R², R² and R³, or R³ and R⁴ forma fused 5- or 6-membered carbocyclic, heterocyclic, aromatic orheteroaromatic ring.

Preferably, at least one of R¹-R⁴ is alkyl, alkoxy, hydroxy, or azido,or R¹ is cyano, or R¹ and R², R² and R³, or R³ and R⁴ form a fused 5- or6membered carbocyclic, heterocyclic, aromatic, or heteroaromatic ring.

The invention also relates to certain compounds found to haveexceptional in vivo activity and having the Formula (III)

or a tautomer thereof;

wherein

R¹ is nitro, fluoro, or chloro;

R² is fluoro, chloro, alkyl, alkoxy, or azido;

R³ is fluoro or chloro; and

R⁴ is hydrogen or fluoro;

with the proviso that at least one of R¹-R⁴ is fluoro, and that R² andR⁴ are not fluoro when R¹ is nitro.

The invention also relates to certain compounds having the Formula IIIor a tautomer thereof, wherein

R¹ is nitro, cyano, CF₃, carboxy, or alkanoyl;

R² is alkoxy, aralkoxy, thioalkyl, carboxy, alkanoyl, hydroxy,mercaptoalkyl, azido, or NR⁵R⁶, wherein R⁵ and R⁶ are independentlyhydrogen, alkyl, or aryl groups;

R³ is halo, haloalkyl, nitro, alkyl, alkoxy, azido, or cyano; and

R⁴ is hydrogen.

The invention also relates to a method for the preparation of a1,4-dihydroquinoxaline-2,3-dione having the Formula (IV):

or a tautomer thereof;

wherein

R¹ is nitro;

R² is haloalkyl, halo, cyano, alkyl, or alkoxy;

R³ is haloalkyl, halo, cyano, alkyl, or alkoxy; and

R⁴ is hydrogen;

comprising reaction of a compound having the Formula (V):

or a tautomer thereof;

wherein

R¹ is hydrogen;

R² is haloalkyl, halo, cyano, alkyl, or alkoxy;

R³ is haloalkyl, halo, cyano, alkyl, or alkoxy; and

R⁴ is hydrogen;

with fuming nitric acid; and isolating the1,4-dihydroquinoxaline-2,3-dione so produced.

The invention also relates to a method for the preparation of a compoundhaving the Formula (VI):

or a tautomer thereof;

wherein

R¹ is nitro, cyano, CF₃, carboxy, or alkanoyl;

R² is alkoxy, aralkoxy, hydroxy, mercaptoalkyl, azido, or NR⁵R⁶, whereinR⁵ and R⁶ are independently hydrogen, alkyl, or aryl groups;

R³ is halo, haloalkyl, nitro, akyl, alkoxy, azido, or cyano; and

R⁴ is hydrogen;

comprising reaction of a compound having the Formula (VII):

or a tautomer thereof;

wherein

R¹ is nitro, cyano, CF₃, carboxy, or alkanoyl;

R² is fluoro;

R³ is halo, haloalkyl, nitro, alkyl, alkoxy, azido, or cyano; and

R⁴ is hydrogen;

with an alkoxide, aryl alkoxide, hydroxide, an all mercaptide, azide, orHNR⁵R⁶ respectively, in an inert solvent, and isolating the compound soproduced.

In one embodiment, the present invention relates to a method of treatingor preventing (A) neuronal loss associated with stroke, ischemia, CNStrauma, or hypoglycemia or (13) the adverse neurological consequences ofsurgery, comprising administering to an animal in need of such treatmentor prevention an effective amount of a compound of the Formula I or atautomer thereof.

In a second embodiment, the present invention relates to a method oftreating a neurodegenerative disease selected from Alzheimer's disease,amyotrophic lateral sclerosis, Huntington's disease, and Down'ssyndrome, comprising administering to an animal in need of suchtreatment an effective amount of a compound of the Formula I or atautomer thereof.

In a third embodiment, the present invention relates to a method ofantagonizing excitatory amino acids at the NMDA receptor complex,comprising administering to an animal in need thereof an effectiveamount of a compound of the Formula I or a tautomer thereof.

In a fourth embodiment, the present invention relates to a method oftreating or preventing the adverse consequences of the hyperactivity ofthe NMDA receptor, comprising administering to an animal in need of suchtreatment or prevention an effective amount of a compound of the FormulaI or a tautomer thereof.

In a fifth embodiment, the present invention relates to a method oftreating chronic pain, comprising administering to an animal in need ofsuch treatment an effective amount of a compound of the Formula I or atautomer thereof.

In a sixth embodiment, the present invention relates to a method oftreating or preventing anxiety, comprising administering to an animal inneed of such treatment or prevention an effective amount of a compoundof the Formula I or a tautomer thereof.

In a seventh embodiment, the present invention relates to a method oftreating or preventing convulsions, comprising administering to ananimal in need of such treatment or prevention an effective amount of acompound of the Formula I or a tautomer thereof.

In an eighth embodiment, the present invention relates to a method ofinducing anesthesia, comprising administering to an animal in need ofsuch anesthesia an effective amount of a compound of the Formula I or atautomer thereof.

In a ninth embodiment, the present invention relates to a method oftreating or preventing NMDA receptor-ion channel related psychosis,comprising administering to an animal in need of such treatment orprevention an effective amount of a compound of the Formula I or atautomer thereof.

In a tenth embodiment, the present invention relates to a method ofinducing a hypnotic effect, comprising administering to an animal inneed of such treatment an effective amount of a compound having theFormula I or a tautomer thereof.

In an eleventh embodiment, the present invention relates to aradiolabelled compound having the Formula I or a tautomer thereof.

In a twelfth embodiment, the present invention relates to apharmaceutically acceptable salt of a compound having the Formula I or atautomer thereof.

In a thirteenth embodiment, the present invention relates to a method ofpreventing opiate tolerance, comprising administering to an animal inneed of such prevention an effective amount of a compound of the Formula

or a tautomer thereof;

wherein

R is hydrogen, hydroxy, amino, —CH₂CONHAr, —NHCONHAr, —NHCOCH₂Ar,—COCH₂Ar, wherein Ar is an aryl group, or a radical having the formula:

wherein R⁶ is hydrogen, lower alkyl of 1-6 carbon atoms, or aryl; R⁷ ishydrogen or lower alkyl of 1-6 carbon atoms; n is an integer from 0 to5; and R⁸ is hydrogen, C₁₋₆ alkyl, or aralkyl;

R¹, R², R³, and R⁴ are independently selected from the group consistingof hydrogen, hydroxy, acyloxy, aralkoxy, amino, alkanoylamino, halo,haloalkyl, nitro, alkyl, alkoxy, carboxy, alkanoyl, thioalkyl,alkoxyalkyl, aryloxyalkyl, arylalkyl, alkenyl, alkynyl, arylalkenyl,arylalkynyl, cyano, cyanomethyl, dicyanomethyl, cyanoamino,dicyanoamino, or azido; or where R¹ and R², R² and R³, or R³ and R⁴ forma fused 5- or 6-membered carbocyclic, heterocyclic, aromatic orheteroaromatic ring.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph showing the effect of5-nitro-6,7-dimethylquinoxalinedione (NDMQX) on the early phase of theformalin test for pain.

FIG. 2 depicts a graph showing the effect of NDMQX on the late phase ofthe formalin test for pain.

FIG. 3 depicts a graph showing the time course of7-chloro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione (NMCQX) {7.5mg/kg (Tris 0.05 M)} in the MES test for anti-convulsant effect.

FIG. 4 depicts a graph showing the dose response of NMCQX, 60 minutesfollowing i.p. injection, in the MES test for anti-convulsant effect.

FIG. 5 depicts a graph showing the time course of NDMQX {15 mg/kg(Arginine 0.1 M)} in the MES test for anti-convulsant.

FIG. 6 depicts a graph showing the dose response of NDMQX, 30 minutesfollowing i.p. injection, in the MES test for anti-convulsant effect.

FIG. 7 depicts a graph showing the effect of chronic (8 days) NDMQX onmorpine tolerance in the formalin test {@, p<0.03; *, p<0.05}.

FIG. 8 depicts a graph showing the effect of chronic (8 days) NDMQX onmorphine-induced antinociception in the formalin test.

FIG. 9 depicts a graph showing the effect of NDMQX on cortical andsubcortical infarct volume.

FIG. 10 depicts a graph showing the effect of NMCQX on cortical andsubcortical infarct volume.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to compounds having a high affinity forthe glycine binding site and the capability of crossing the blood brainbarrier at high levels, while lacking PCP side effects. Such compoundsinclude alkyl, azido, alkoxy, and fluoro-substituted1,4-dihydroquinoxaline-2,3-diones, which are highly selective,competitive antagonists of the glycine binding site of the NMDAreceptor. Certain of the 1,4-dihydroquinoxaline-2,3-diones of theinvention have the following Formula (I):

or a tautomer thereof;

wherein

R is hydrogen, hydroxy, amino, —CH₂CONHAr, —NHCONHAr, —NHCOCH₂Ar,—COCH₂Ar, wherein Ar is an aryl group, or a radical having the formula:

wherein R⁶ is hydrogen, lower alkyl of 1-6 carbon atoms, or aryl; R⁷ ishydrogen or lower alkyl of 1-6 carbon atoms; n is an integer from 0 to5; and R⁸ is hydrogen, C₁₋₆ alkyl, or aralkyl;

R¹, R², R³, and R⁴ are independently selected from the group consistingof hydrogen, hydroxy, acyloxy, aralkoxy, amino, alkanoylamino, halo,haloalkyl, nitro, alkyl, alkoxy, carboxy, alkanoyl, thioalkyl,alkoxyalkyl, aryloxyalkyl, arylalkyl, alkenyl, alkynyl, arylalkenyl,arylalkynyl, cyano, cyanomethyl, dicyanomethyl, cyanoamino,dicyanoamino, or azido;

or where R¹ and R², R² and R³, or R³ and R⁴ form a fused 5- or6-membered carbocyclic, heterocyclic, aromatic, or heteroaromatic ring;

with the proviso that at least one of R¹-R⁴ is alkyl, alkoxy, aralkoxy,hydroxy, carboxy, alkanoyl, or azido, or that R¹ is cyano, or that R¹and R², R² and R³, or R³ and R⁴ form a 5- or 6-membered carbocycloalkyl,heterocyclic, aromatic, or heteroaromatic ring.

Of course, it is to be understood that R¹-R⁴ can be the same ordifferent.

In preferred compounds within the scope of Formula I, R¹ is alkyl,azido, alkoxy, hydroxy, haloakyl, halo, nitro, cyano, or alkanoylamino;R² is alkyl, azido, alkoxy, aralkoxy, cyano, haloalkyl, halo, hydroxy,or nitro; R³ is alkyl, azido, alkoxy, cyano, halo, haloalkyl, orhydroxy, and R⁴ is alkyl, azido, alkoxy, cyano, hydroxy, or hydrogen;but at least one of R¹-R⁴ is alkyl, alkoxy, hydroxy, or azido, or R¹ iscyano. Especially preferred compounds are those where R¹ is alkyl,azido, alkoxy, cyano, hydroxy, or nitro; R² is alkyl, azido, alkoxy,aralkoxy, cyano, hydroxy, or halo; R³ is alky, azido, alkoxy, halo,cyano, hydroxy, or haloalkyl; and R⁴ is alkyl, alkoxy, azido, cyano,hydroxy, or hydrogen; but where at least one of R¹-R⁴ is alkyl, alkoxy,hydroxy, or azido, or R¹ is cyano. In general, the most preferredcompounds are substituted by hydrogen or fluorine at position R⁴ when Ris other than hydrogen. When R is hydrogen, the most preferred compoundsare those where R⁴ is hydrogen or fluoro and R¹-R³ are other thanhydrogen.

Other preferred compounds are substituted by an arylalkyl, arylalkenyl,arylalkynyl, or aryloxyalkyl group in the 6-position of the 1,4dihydroquinoxaline-2,3-dione ring. Such compounds are expected to showincreased lipophilicity and, therefore, ability to cross the blood-brainbarrier. Such compounds are also expected to bind favorably to ahypothetical hydrophobic binding pocket that might be present at aposition 10 o'clock to the 1,4-dihydroquinoxaline-2,3-dione ring.Alternatively, a long chain alkanoylamido group (an acylamino group) canbe present at the 5-position of the ring to interact with this bindingpocket.

Where the 1,4-dihydroquinoxaline-2,3-dione is substituted by a radicalhaving Formula II, the radical can be a C₂₋₇carboxyalkyl group includingcarboxymethyl, 2-carboxyethyl, 3-carboxypropyl, 4-carboxybutyl,5-carboxypentyl, 6-carboxyhexyl, 1-carboxyethyl, 1-carboxypropyl,1-carboxybutyl, 1-carboxypentyl, 1-carboxyhexyl, 2-carboxypropyl,2-carboxybutyl, 2-carboxypentyl, 2-carboxyhexyl, 3-carboxybutyl,3-carboxypentyl, 3-carboxyhexyl, 5-carboxypentyl, 5-carboxyhexyl, andthe like.

Typical C₈₋₁₂ carboxyaralkyl groups that are included in Formula IIinclude 1-aryl-2-carboxyethyl, 1-aryl-3-carboxypropyl,1-aryl-4-carboxybutyl, 1-aryl-5-carboxypentyl, 1-aryl-6-carboxyhexyl,1-aryl-1-carboxyethyl, 1-aryl-1-carboxypropyl, 1-aryl-1-carboxybutyl,1-aryl-1-carboxypentyl, 1-aryl-1-carboxyhexyl, 1-aryl-2-carboxypropyl,1-aryl-2-carboxybutyl, 1-aryl-2-carboxypentyl, 1-aryl-2-carboxyhexyl,1-aryl-3-carboxybutyl, 1-aryl-3-carboxypentyl, 1-aryl-3-carboxyhexyl,1-aryl-5-carboxypentyl, 1-aryl-5-carboxyhexyl, 2-aryl-2-carboxyethyl,2-aryl-3-carboxypropyl, 2-aryl-4-carboxybutyl, 2-aryl-5-carboxypentyl,2-aryl-6-carboxyhexyl, 2-aryl-1-carboxyethyl, 2-aryl-1-carboxypropyl,2-aryl-1-carboxybutyl, 2-aryl-1-carboxypentyl, 2-aryl-1-carboxyhexyl,2-aryl-2-carboxypropyl, 2-aryl-2-carboxybutyl, 2-aryl-2-carboxypentyl,2-aryl-2-carboxyhexyl, 2-aryl-3-carboxybutyl, 2-aryl-3-carboxypentyl,2-aryl-3-carboxyhexyl, 2-aryl-5-carboxypentyl, 2-aryl-5-carboxyhexyl,and the like.

Typical C₁₋₆ alkyl groups include methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert.-butyl, pentyl, 2-pentyl, 3-pentyl,neopentyl, hexyl, 2-hexyl, 3-hexyl, 2-methyl-1-pentyl,3-methyl-1-pentyl, 4-methyl-1-pentyl, and the like.

Typical alkoxy groups include oxygen substituted by any of the C₁₋₆alkylgroups mentioned above.

Typical thioalkyl groups include sulfur substituted by any of theC₁₋₆alkyl groups mentioned above.

Typical alkoxyalkyl groups include any of the above alkyl groupssubstituted by an alkoxy group, such as methoxymethyl, ethoxymethyl,propoxymethyl, butoxymethyl, pentoxymethyl, hexoymethyl, methoxyethyl,methoxypropyl, methoxybutyl, methoxypentyl, methoxyhexyl, and the like.

Preferred aryl groups are C₆₋₁₄ aryl groups and typically includephenyl, naphthyl, fluorenyl, phenanthryl, and anthracyl groups.

Typical aralkoxy groups include the above alkoxy groups substituted byone or more of the above aryl groups, e.g., 3-phenylpropoxy,2-phenylethoxy, and the like.

Typical aryloxyalkyl groups include any of the above alkyl groupssubstituted by an aryloxy group, such as, phenoxymethyl, phenoxyethyl,phenoxypropyl, phenoxybutyl, phenoxypentyl, phenoxyhexyl, and the like.

Typical arylalkyl groups include any of the above C₁₋₆ alkyl groupssubstituted by any of the C₆₋₁₄ aryl groups, including the groupPh(CH₂)_(n), where n is 1-6, for example, benzyl, 2-phenethyl,3-phenylpropyl, 2-phenylpropyl, 4-phenylbutyl, 5-phenylpentyl, and6-phenylhexyl groups.

Typical C₂₋₆ alkenyl groups include ethenyl, 2-propenyl, isopropenyl,2-butenyl, 3-butenyl, 4-pentenyl, 3-pentenyl, 2-pentenyl, 5-hexenyl,4-hexenyl, 3-hexenyl, and 2-hexenyl groups.

Typical C₂₋₆ alkynyl groups include ethynyl, 2-propynyl, 2-butynyl,3-butynyl, 4-pentynyl, 3-pentynyl, 2-pentynyl, 5-hexynyl, 4-hexynyl,3-hexynyl, and 2-hexynyl groups.

Typical arylalkenyl groups include any of the above C₂₋₆ alkenyl groupssubstituted by any of the above C₆₋₁₄ aryl groups, e.g. 2-phenylethenyl,3-phenyl-2-propenyl, 2-phenylisopropenyl, 4-phenyl-2-butenyl,4-phenyl-3-butenyl, 5-phenyl-4-pentenyl, 5-phenyl-3-pentenyl,5-phenyl-2-pentenyl, 6-phenyl-5-hexenyl, 6-phenyl-4-hexenyl,6-phenyl-3-hexenyl, and 6-phenyl-2-hexenyl groups.

Typical arylalkynyl groups include any of the above C₂₋₆ alkynyl groupssubstituted by any of the above C₆₋₁₄ aryl groups, e.g. 2-phenylethynyl,2-phenyl-2-propynyl, 4-phenyl-2-butynyl, 4-phenyl-3-butynyl,5-phenyl-4-pentynyl, 5-phenyl-3-pentynyl, 5-phenyl-2-pentynyl,6-phenyl-5-hexynyl, 6-phenyl-4-hexynyl, 6-phenyl-3-hexynyl, and6-phenyl-2-hexynyl groups.

Typical halo groups include fluorine, chlorine, bromine, and iodine.

Typical haloalkyl groups include C₁alkyl groups substituted by one ormore fluorine, chlorine, bromine, or iodine atoms, e.g. fluoromethyl,difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl,and trichloromethyl groups.

Typical amino groups include —NH₂, NHR⁹, and —NR⁹R¹⁰, wherein each of R⁹and R¹⁰ is one of the C₁₋₆ alkyl groups mentioned above. The amino groupcan also be substituted with one (—NHCN) or two (—N(CN)₂) cyano groups.These groups can be prepared by the reaction of the corresponding aminewith cyanogen bromide.

Typical alkanoyl groups include C₁₋₅C(O) allnyl groups, e.g. acetyl,propionyl, butanoyl, pentanoyl, and hexanoyl groups, or by anarylalkanoyl group, e.g., a C₁₋₅C(O) alkanoyl group substituted by anyof the above aryl groups.

As used herein, the term “acyloxy” means an alkanoyl group attached tothe ring via an oxygen atom, e.g., acetyloxy, 2-phenylacetyloxy, and thelike.

Typical alkanoylamino groups include an amino group substituted by oneof the Cl₁₋₅C(O) alkanoyl groups.

Typical fused ring systems where R¹ and R², R² and R³, or R³ and R⁴ arelinked together to form a 5- or 6-membered carbocyloalkyl, heterocyclic,aromatic, or heteroaromatic group include benzo[g]quinoxaline,cyclohexo[g]quinoxaline, cyclopento[g]quinoxaline, thieno[g]quinoxaline,tetrahydrothieno[g]quinoxaline, furo[g]quinoxaline,tetrahydrofuro[g]quinoxaline, pyrano[g]quinoxaline,pyrrolo[g]quinoxaline, pyrido[g]quinoxaline, pyrazino[g]quinoxalinepyridazino[g]quinoxaline,6,7-methylenedioxy-1,4-dihydroquinoxaline-2,3-dione, and 6,7-cycliccarbonate 1,4-dihydroquinoxaline-2,3-dione. The fused heterocyclic ringcan bear a carbonyl group giving a fused lactone or lactam group.

Particularly preferred substituted 1,4-dihydroquinoxaline-2,3-diones ofthe present invention include, but are not limited to,6,7-dimethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6,7-dimethyl-1,4-dihydroquinoxaline-2,3-dione;7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;5,7-dimethyl-6-nitro-1,4-dihydroquinoxaline-2,3-dione;6,7-diethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-bromo-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-fluoro-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6,7-dimethoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-azido-5,7-difluoro-1,4-dihydroquinoxaline-2,3-dione;6-azido-5,7-dichloro-1,4-dihydroquinoxaline-2,3-dione;5-azido-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione;6,7-dimethyl-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;5,7-dimethyl-1,4-dihydroquinoxaline-2,3-dione;7-methyl-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;6,7-diethyl-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;5,7-diethyl-6-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;6-bromo-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-iodo-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-iodo-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-6-methoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-methoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-azido-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-methyl-5,6-dinitro-1,4-dihydroquinoxaline-2,3-dione;6-methyl-5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione;6,7-dichloro-5-cyano-1,4-dihydroquinoxaline-2,3-dione;6,7-difluoro-5-cyano-1,4-dihydroquinoxaline-2,3-dione;1,4-dihydrobenzo-[g]quinoxaline-2,3-dione;1,4-dihydro-5-nitrobenzo[g]quinoxaline-2,3-dione;1,4-dihydro-5-nitrocyclopento[g]quinoxaline-2,3-dione;1,4-dihydro-5-nitrocyclohexo[g]quinoxaline-2,3-dione;1,4-dihydro-5-nitropyrrolo[2,3-g]quinoxaline-2,3-dione;1,4-dihydro-5-nitropyrrolo[3,2-g]quinoxaline-2,3-dione;5,6,7,8-tetrafluoro-1,4-dihydroquinoxaline-2,3-dione;5-chloro-7-fluoro-1,4-dihydroquinoxaline-2,3-dione;5-bromo-7-fluoro-1,4-dihydroquinoxaline-2,3-dione;4-carboxymethyl-5-chloro-6,7-difluoro-1,4-dihydroquinoaine-2,3-dione;4-carboxymethyl-5-bromo-6,7-difluoro-1,4-dihydroquinoxaline-2,3-dione;4-carboxymethyl-5,6,7,8-tetrafluoro-1,4-dihydroquinoxaline-2,3-dione;4-carboxymethyl-5-chloro-7-fluoro-1,4-dihydroquinoxaline-2,3-dione;4-carboxymethyl-5-bromo-7-fluoro-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-6-nitro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;6-amino-7-fluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;6,7,8-trifluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6,7,8-trifluoro-5-chloro-1,4-dihydroquinoxaline-2,3-dione;6,7,8-trifluoro-5-bromo-1,4-dihydroquinoxaline-2,3-dione;6,7,8-trifluoro-5-iodo-1,4-dihydroquinoxaline-2,3-dione;6,7,8-trifluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;5,6,7-trifluoro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-5,7-difluoro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6,8-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6,8-difluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;7-chloro-5,6-difluoro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-7,8-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-7,8-difluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;6,7-dichloro-8-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6,7-dichloro-8-fluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;5,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;7-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-fluoro-5,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;6-chloro-5,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;6-nitro-5,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;6-bromo-5,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;6-iodo-5,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;6,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;5-nitro-6,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;5-chloro-6,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;5-fluoro-6,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;5-bromo-6,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;5-iodo-6,7-bis(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;5,6,7-tris(trifluoromethyl)-1,4-dihydroquinoxaline-2,3-dione;6,7-dichloro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;6,7-difluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-bromo-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-bromo-6-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-bromo-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-6-iodo-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-fluoro-7-iodo-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-6-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-fluoro-7-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-bromo-7-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-bromo-6-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-iodo-7-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-iodo-6-trifluoromethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-fluoro-5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-5,6-dinitro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione;7-bromo-5,6-dinitro-1,4-dihydroquinoxaline-2,3-dione;6-bromo-5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione;7-iodo-5,6-dinitro-1,4-dihydroquinoxaline-2,3-dione;6-iodo-5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione;7-trifluoromethyl-5,6-dinitro-1,4-dihydroquinoxaline-2,3-dione;6-trifluoromethyl-5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione;5-amino-7-chloro-6-methyl-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-ethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;5-chloro-8-methyl-1,4-dihydroquinoxaline-2,3-dione;5-chloro-8-methyl-6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-7-ethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-7-ethyl-1,4-dihydroquinoxaline-2,3-dione;6-chloro-7-ethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-ethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione;6-chloro-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-ethylthio-5-nitro-1,4-dihydroquinoxaline-2,3-dione;5-hydroxy-6,7-dimethoxy-1,4-dihydroquinoxaline-2,3-dione;5-acetyloxy-6,7-dimethoxy-1,4-dihydroquinoxaline-2,3-dione;5-(2-phenylacetyloxy)-6,7-dimethoxy-1,4-dihydroquinoxaline-2,3-dione;5,6,7-trihydroxy-1,4-dihydroquinoxaline-2,3-dione;6,7-dihydroxy-1,4-dihydroquinoxaline-2,3-dione;6-(n-butoxy)-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione; and7-fluoro-5-nitro-6-(3-phenylpropoxy)-1,4-dihydroquinoxaline-2,3-dione.

The present invention relates in part to the discovery that certainalkyl substituted quinoxaline-2,3-diones have high affinity for theglycine/NMDA receptor and have unexpectedly high in vivo activity asanticonvulsants in maximum electroshock seizure (MES) experiments inmice. Therefore, these compounds are able to cross the blood brainbarrier at high levels.6,7-Dimethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione (NDMQX) was foundto have high affinity for the glycine/NMDA receptor with K_(i) of 43 nM,which is about 10 times less active than6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione. NDMQX, however,was found to have unexpectedly high in vivo activity. It has an ED₅₀ of4-5 mg/kg as an anticonvulsant in the MES experiment in mice. Incomparison, 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione has anED₅₀ of 4-5 mg/kg as an anticonvulsant in the MES experiment in mice.Thus, NDMQX appears to be 10 times better in crossing the blood brainbarrier than 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione.

Since the methyl groups in NDMQX could be replaced by other longer alkylgroups, such as, ethyl or propyl, or by arylalkyl groups, such as,benzyl and phenethyl, as well as by longer alkyl chains containing ethergroups, such as, methoxyethyl, which are expected to increase thelipophilicity of the molecule and increase the ability to cross theblood brain barrier, the above discovery has led to a new group ofquinoxalinediones (QXs) with good in vivo properties. The R² and R³groups in the 6 and 7 positions, respectively, can also be incorporatedinto a ring system, as shown in Formula VIII. It could be a saturatedsystem and contain hetero atoms, or an unsaturated system, such as,1,4-dihydrobenzo[g]quinoxaline-2,3-dione (IX).

Another group of highly interesting compounds are those substituted withboth alkyl and halogen substituents, such as,6-chloro-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-fluoro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione, and7-chloro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione, which areexpected to combine the best of6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione,6,7-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione, and6,7-dimethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione, having highaffinity for the glycine/NMDA receptor and being able to cross the bloodbrain barrier at high levels.7-Chloro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione (NMCQX) wasfound to have a high affinity for the glycine/NMDA receptor with a K_(i)of 5 nM, which is about as good as6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione. NMCQX, however,was found to have unexpectedly high in vivo activity. It had an ED₅₀ of1 mg/kg as an anticonvulsant in the MES experiment in mice, which isabout 4-5 times better than6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione (ED₅₀=4-5 mg/kg).7-Fluoro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione (K_(i)=53 nM,ED₅₀=2 mg/kg) and6-chloro-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione (K_(i)=27 nM,ED₅₀₌₂ mg/kg) also were found to have a high affinity for theglycine/NMDA receptor and to have unexpectedly high in vivo activity.

The present invention also relates to the discovery that certainfluoro-substituted 1,4-dihydroquinoxaline-2,3-diones have a highaffinity for the glycine/NMDA receptor and have unexpectedly high invivo activity as anticonvulsants in the MES experiment in mice (TableIII). Therefore, these compounds are able to cross the blood brainbarrier at high levels.6,7-Difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione was found to havea high affinity for the glycine/NMDA receptor with a K_(i) of 87 nM,which is more than 20 times less active than6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione (K_(i)=3.3 nM).6,7-Difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione, however, wasfound to have surprisingly high in vivo activity. It has an ED₅₀ of0.7-0.8 mg/kg as an anticonvulsant in the MES experiment in mice. Incomparison, 6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione has anED₅₀ of 4-5 mg/kg as an anticonvulsant in the MES experiment in mice.This means that 6,7-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dionemight be about 100 times better in crossing the blood brain barrier than6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione.

In general, compounds that are used for treating animals should not havea fluorine in the 6- or 8- positions and an electron withdrawing group,such as, nitro, in the 5-position, as such compounds are unstable. Asdiscussed herein, the fluorine group in such compounds is readilydisplaced by common nucleophiles. Thus, although6,7-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione has good in vivoactivity, it should not be administered to animals, as it may react withbiological nucleophiles.

7-Chloro-6,8-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione was foundto have high affinity for the glycine/NMDA receptor with K_(i) of 170nM, which is about 50 times less active than6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione.7-Chloro-6,8-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione also wasfound to have surprisingly high in vivo activity. It has an ED₅₀ of 2-3mg/kg as an anticonvulsant in the MES experiment in mice. In comparison,6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione has an ED₅₀ of 4-5mg/kg as an anticonvulsant in the MES experiment in mice. This meansthat 7-chloro-6,8-difluoro-5-nitro-1,4 dihydroquinoxaline-2,3-dionemight be about 100 times better in crossing the blood brain barrier than6,7-dichloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione.

The compounds disclosed herein are active in treating or preventingneuronal loss, neurodegenerative diseases, and chronic pain and areactive as anticonvulsants and in inducing anesthesia without untowardside effects caused by non-selective binding with other receptors,particularly, kainate, AMPA, and quisqualate receptors and the PCP andglutamate receptors associated with the NMDA receptor. In addition,these compounds are effective in treating or preventing the adverseconsequences of the hyperactivity of the excitatory amino acids, e.g.,those that are involved in the NMDA receptor system, by blocking theglycine receptors and preventing the ligand-gated cation channels fromopening and allowing excessive influx of Ca⁺⁺ into neurons, as occursduring ischemia.

Neurodegenerative diseases that may be treated with the disclosedcompounds include those selected from the group consisting ofAlzheimer's disease, amyotrophic lateral sclerosis, Huntington'sdisease, and Down's syndrome.

These compounds also find particular utility in the treatment orprevention of neuronal loss associated with multiple strokes that giverise to dementia. After a patient has been diagnosed as suffering from astroke, the compounds can be administered to ameliorate the immediateischemia and prevent further neuronal damage that may occur fromrecurrent strokes.

Moreover, these compounds are able to cross the blood/brain barrier, incontrast to 6-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione,6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione, and other6,7-disubstituted 1,4-dihydroquinoxaline-2,3-diones that are incapableof crossing the blood/brain barrier after i.p. administration (seeTurski, L. et al., J. Pharm. Exp. Ther. 260: 742-747 (1992)). See also,Sheardown et al., Eur. J. Pharmacol. 174:197-204(1989), who disclosethat 5,7-dinitro-1,4-dihydroquinoxaline-2,3-dione,6,7-dinitro-1,4-dihydroquinoxaline-2,3-dione, andcyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione have poor access to thecentral nervous system.

For a compound to begin to show in vivo efficacy and, thus, the abilityto cross the blood-brain barrier, the compound should exhibit an ED₅₀ ofless than about 100 mg/kg body weight of the animal. Preferably, thecompounds of the present invention exhibit an ED₅₀ of less than about 20mg/kg and, more preferably, less than about 10 mg/kg.

These compounds find particular utility in treating or preventing theadverse neurological consequences of surgery. For example, coronarybypass surgery requires the use of heart-lung machines, which tend tointroduce air bubbles into the circulatory system that may lodge in thebrain. The presence of such air bubbles robs neuronal tissue of oxygen,resulting in anoxia and ischemia. Pre- or post-surgical administrationof the 1,4-dihydroquinoxalines of the present invention will treat orprevent the resulting ischemia. In a preferred embodiment, the compoundsare administered to patients undergoing cardiopulmonary bypass surgeryor carotid endarterectomy surgery.

These compounds also find utility in treating or preventing pain, e.g.,chronic pain. Such chronic pain can be the result of surgery, trauma,headache, arthritis, or other degenerative disease. The compounds of thepresent invention find particular utility in the treatment of phantompain that results from amputation of an extremity. In addition totreatment of pain, the compounds of the invention are also useful ininducing anesthesia, either general or local anesthesia, as, forexample, during surgery.

The compounds of the present invention can be tested for potentialglycine antagonist activity by observing the inhibition of binding of 1μM glycine-stimulated [³H]-MK-801 in rat or guinea pig brain membranehomogenates. The more potent the glycine antagonist, the less[³H]-MK-801 can bind since the [³H]-MK801 binding site (PCP receptor) isaccessible only upon the opening of the ion channel by glutamate andglycine (Fletcher, E. L., et al., in Glycine Neurotransmission,Otterson, P., et al. (eds.), John Wiley and Sons (1990); Johnson, J. W.,et at., Nature 325:529 (1987)).

The binding affinities of quinoxaline-2,3-diones at NMDA receptorglycine sites also were estimated by electrophysiological assays witheither cloned rat NMDA receptors expressed in Xenopus oocytes, ornon-NMDA receptors expressed in oocytes by whole rat brain poly(A)⁺ RNA.K_(i) values were estimated by assuming competitive inhibition andassaying suppression of membrane current responses elicited by fixedconcentrations of agonist: 1 mM glycine and 100 mM glutamate for NMDAreceptors; 20 mM kainic acid for non-NMDA receptors. For NMDA receptorsK_(i) values were approximated by averaging values at three subtypecombinations (NR1A/NR2A, NR1A/NR2B, and NR1A/NR2C). See U.S. applicationSer. No. 08/148,259, entitled Glycine Receptor Antagonists and the UseThereof, supra.

Preferably, the compounds of the invention exhibit a binding affinity tothe glycine binding site of K_(i)=about 10 μM or less, more preferably,1 μM or less, and more preferably, 500 nM or less, and more preferably,100 nM or less, and most preferably, about 10 nM or less. Alsopreferable are compounds that exhibit binding at the kainate and AMPAsites of not less than K_(i)=1 μM and, more preferably, not less than 10μM.

The novel glycine antagonists can be tested for in vivo activity afterintraperitoneal injection using a number of anticonvulsant tests in mice(audiogenic seizure model in DBA-2 mice, pentylenetetrazol-inducedseizures in mice, NMDA-induced death in mice, and MES in mice).Preferred compounds exhibit ataxia side effects in the rotorod ataxiatest at dosage levels of greater than about 100 mg/kg, more preferably,greater than about 200 mg/kg.

The compounds can also be tested in drug discrimination tests in ratstrained to discriminate PCP from saline. It is expected that most of thecompounds will not generalize to PCP at any dose. In addition, it isalso expected that none of the compounds will produce a behavioralexcitation in locomotor activity tests in the mouse. It is expected thatsuch results will suggest that the glycine, AMPA, kainate, andquisqualate antagonists of the present invention do not show thePCP-like behavioral side effects that are common to NMDA channelblockers such as MK-801 and PCP or to competitive NMDA antagonists suchas CGS19755.

The glycine and excitatory amino acid antagonists are also expected toshow potent activity in vivo after intraperitoneal injection suggestingthat these compounds can penetrate the blood/brain barrier.

Azido-substituted 1,4-dihydroquinoxaline-2,3-diones can be employed tophotoaffinity-label the glycine receptor. Unexpectedly, it has beendiscovered that 6-azidol-1,4-dihydroquinoxaline-2,3-dione quitefavorably binds to the glycine binding site, as compared to thecorresponding 6-CF₃,6-NO₂, 6-F, 6-CN, 6-NH₂, and unsubstitutedderivatives (Table I):

TABLE I

K_(i) R² (nM) N₃  210 CF₃  360 NO₂ 1395 H 2466 F 2880 CN 4889 NH₂inactive

The compounds of the present invention can be prepared as follows. Asshown in Scheme I, the isomeric6-halo-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione and7-halo-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione can be preparedfrom a 3-halo-4-methylaniline by protection of the amino group with, forexample, trifluoroacetic acid anhydride, followed by ortho nitration.Removal of the amino-protecting group and reduction of the orthonitrogroup gives the 3-halo-4-methyl-1,2-phenylenediamine. This1,2-phenylenediamine can then be condensed with oxalic acid to give the7-halo-6-methyl-1,4-dihydroquinoxaline-2,3-dione. Nitration leads to amixture of the two isomeric compounds.

Alternatively, the intermediate trifluoroacetyl2-amino-5-halo-4-methylanilide can be further treated withtrifluoroacetic acid anhydride followed by nitration to give twoisomeric nitro compounds, which may be separated. Removal of theprotecting groups, condensation with oxalic acid, and subsequentnitration gives the isomerically pure7-halo-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione and6-halo-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione. (See SchemeII).

A second approach was based on the discovery that nitration of3,4-dihydroquinoxaline-2(1H)-one resulted exclusively in the productwith the nitro group in the 5-position. For example, treatment of2,5-difluoro-4-nitrotoluene with sodium glycinate gave the substitutedaniline. The nitro group was reduced by SnCl₂ and the productspontaneously cyclized to give the 3,4-dihydroquinoxaline-2(1H)-one. Itwas nitrated by fuming HNO₃ in trifluoroacetic acid, resulting in bothnitration and oxidation of the compound to give7-fluoro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione in a singlestep (Scheme III. The F—H coupling constants measured from the ¹H NMRspectrum confirmed that the fluoro was meta to the nitro group.

Similarly, treatment of 1-bromo-2,4-difluoro-5-nitrobenzene with sodiumglycinate gave a mixture of the substituted anilines. The nitro groupwas reduced by SnCl₂ and the product spontaneously cyclized to give the3,4-dihydroquinoxaline-2(1H)-one. It was nitrated by HNO₃ intrifluoroacetic acid, resulting in both nitration and oxidation of thecompound to give7-bromo-6-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione in a singlestep (Scheme MV). The F—H coupling constants measured from the ¹H NMRspectrum confirmed that the fluoro was ortho to the nitro group.Alternatively, 1-bromo-2,5-fluoro-4-nitrobenzene can be reduced,cyclized, and oxidized with HNO₃ to give6-bromo-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione.

Thus, the invention also relates to a method for the preparation of a1,4-dihydroquinoxaline-2,3-dione having the Formula:

or a tautomer thereof;

wherein

R¹ is nitro;

R² is haloalkyl, halo, cyano, alky, or alkoxy;

R³ is haloalkyl, halo, cyano, alkyl, or alkoxy; and

R⁴ is hydrogen;

comprising reaction of a compound having the Formula:

or a tautomer thereof;

wherein

R¹ is hydrogen;

R² is haloalkyl, halo, cyano, alkyl, or alkoxy;

R³ is haloalkyl, halo, cyano, alkyl, or alkoxy; and

R⁴ is hydrogen;

with fuming nitric acid; and isolating the1,4-dihydroquinoxaline-2,3-dione so produced. A preferred solvent thatcan be used for this reaction is trifluoroacetic acid. The reaction iscarried out at room temperature until there is an absence of startingmaterial (e.g., overnight).

6,7-Dimethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione (NDMQX) can beprepared by condensation of 4,5-dimethyl-1,2-phenylenediamine withoxalic acid followed by nitration (HNO₃/HSO₄ or KNO₃CF₃CO₂H). It wasfound that nitration in trifluoroacetic acid gave a purer product thanin sulfuric acid. (See Scheme V).

Using a procedure similar to that set forth in Scheme I, compoundshaving fused carbocycloalkyl, heteroalkyl, aromatic, and heteroaromaticgroups fused to the 6- and 7-positions of the quinoxaline ring can beprepared. (See Scheme VI).

6,7-Dimethoxy-1,4-dihydroquinoxaline-2,3-dione was prepared from1,2-dimethoxybenzene. Nitration of 1,2-dimethoxybenzene gave1,2-dimethoxy-4,5-dinitrobenzene, which was reduced to1,2-diamino-4,5-dimethoxybenzene. Condensation of the diamine withoxalic acid gave 6,7-dimethoxy-1,4-dihydroquinoxaline-2,3-dione.

5-Nitrocyclopento[g]-1,4-dihydroquinoxaline-2,3-dione was prepared from5-aminoindan as shown in Scheme VII. Nitration of 5-acetamidoindan gavea mixture of nitro products, which was separated by chromatography togive 5-acetamido-6-nitroindan in about 20% yield. Deprotection followedby reduction and condensation of the resulting diamine with oxalic acidgave cyclopento[g]-1,4-dihydroquinoxaline-2,3-dione in good yield.Nitration under conditions of KNO₃/H₂SO₄ or HNO₃/H₂SO₄ gave complicatedproducts. However, nitration under the mild condition of KNO_(3/CF)₃CO₂H gave 5-nitro-cyclopento[g]-1,4-dihydroquinoxaline-2,3-dione as theonly product.

Azido substituted quinoxaline-2,3-diones were prepared by diazotizationof amino substituted 1,4-dihydroquinoxaline-2,3-diones followed bytreatment with sodium azide:

The structure-activity relationships of a number of alkyl, azido,fluoro, it alkoxy, and cyano-substituted1,4-dihydroquinoxaline-2,3-diones are set forth in Table II.

TABLE II Structure and Activity of Alkyl, Azido, Fluoro, Alkoxy, andCyano-Substituted 1,4-Dihydroquinoxaline-2,3-diones

K_(i) R₅ R₆ R₇ R₈ (nM)^(a) NO₂ Me Me H  43 NO₂ Me F H  53 NO₂ Me Br H  40^(b) NO₂ Cl Me H  27 NO₂ Me Cl H  5 NO₂ OMe F H 320 NO₂ OEt F H1000  NO₂ N₃ F H 280 NO₂ OMe Cl H  450^(b) NO₂ Et Cl H  200^(b) NO₂ ClEt H   40^(b) NO₂ OBu-n F H 6500^(b) NO₂ O(CH₂)₃Ph F H 6700^(b) NO₂ SEtCl H 500 NH₂ Me Cl H  500^(b) H OMe OMe H   PA^(b,c) H Et Et H  800^(b)H Cl Et H  730^(b) CN Cl NO₂ H  50 CN Cl Cl H   15^(d) NO₂ H Me H 1200 Me H Me H 1700  Et H Br H 900 H Me Me H 980 Me H H H   PA^(b,c) H Me H H9000^(b) H CH₂CH₂CH₂ H 6300  NO₂ CH₂CH₂CH₂ H 800 H CH═CHCH═CH H 11000 N₃ Cl Cl H  71 Cl N₃ Cl H 536 Cl H H Me   PA^(b,c) Cl NO₂ NO₂ Me3800^(b) H N₃ H H  910^(b) ^(a)From electrophysiology using Xenopusoocytes unless otherwise noted. ^(b)From binding assays. ^(c)PA =partially active. ^(d)Contains ˜4% of the compound in which R₅ = CN, R₆= Cl, R₇ = NH₂, R₈ = H.

Table III sets forth the in vivo activities of a number offluoro-substituted 1,4-dihydroquinoxaline-2,3-diones. As can be seen,these compounds exhibit good anticonvulsant activity in vivo.

Table IV summarizes results of eight 1,4-dihydroquinoxaline-2,3-dionestested i.v. as anticonvulsants in MES experiments in mice. Most of thecompounds tested have very fast peaks of action, especially the fluorosubstituted compounds. The protecting effect of fluoro substitutedcompounds against MES decreased very quickly. After 60 min., no moreprotection was observed for6,7-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione and5,6,7-trifluoro-1,4-dihydroquinoxaline-2,3-dione. Chloro substitutedcompounds also have a fast peak of action, but the protecting effectlasts much longer than fluoro substituted compounds. Trifluoromethylsubstituted compounds have relatively slow peaks of action, and theprotecting effect lasts longer than fluoro substituted compounds. Thisdifferent pattern of action of 1,4-dihydroquinoxaline-2,3-diones couldbe used to apply individual compounds for different types of therapeutictreatment. The present invention is also directed to this discovery.

TABLE III In Vivo Activity of Fluoro Substituted1,4-Dihydroquinoxaline-2,3-diones

ED₅₀ ED₅₀ K_(i) (DBA-2)^(a) (MES)^(c) R₅ R₆ R₇ R₈ (nM) mg/kg mg/kg F F FF 2186   9 20^(a) NO₂ F Cl F 170  ND^(b) 2-3 NO₂ Br F H  43 ND   3.5 NO₂F Cl H  70 ND   0.9 NO₂ Cl F H 180 ND 4-5 F F F H 2966  ND 4-5 NO₂(H) ClF H(NO₂)  18 ND 0.5-0.7 Br F F H 2300  30 ND F Cl F H 1000  ND 10 H F FH 8200  ND 15 NO₂ Me F H  53 ND  2 NO₂ OMe F H 320 ND   7.5 NO₂ N₃ F H280 ND   7.5 NO₂ F F H  87 ND 0.7-0.8 ^(a)i.p. injection. ^(b)ND, notdetermined. ^(c)i.v. injection unless otherwise noted.

TABLE IV 1,4-Dihydroquinoxaline-2,3-diones Tested i.v. for ProtectionAgainst MES

IV peak ED₅₀ ED₁₀₀ K_(i) of action (MES) (MES) % protection R₅ R₆ R₇ R₈(nM) (min) mg/kg mg/kg after 60 min. NO₂ Cl Cl H    3.3  5 4-5  7-10 55(10 mg/kg) NO₂ F Cl F 170 2-5 2-3  5 50 (5 mg/kg)  F F F H 2966   2 4-515-20  0 (10 mg/kg) NO₂(H) Cl F H(NO₂)  18  5 0.5-0.7  4 14 (10 mg/kg)CF₃ H Cl H 395 30 10 30 87 (30 mg/kg) Cl H CF₃ H 320 15 10 20-25 62 (20mg/kg) NO₂ H CF₃ H  95 30 17 40 62 (40 mg/kg) NO₂ F F H  87  1 0.7-0.8  2.5   0 (2.5 mg/kg)

It was discovered during the formulation and stability experiments withnitro and fluoro substituted 1,4-dihydroquinoxaline-2,3-diones that someof these compounds are unstable toward nucleophiles such as thiol (SH)in 2-mercaptoethanol and amino and guanidine in arginine, andreplacement of the F by thiol or amino groups was observed. It wasrecognized that these observations could be utilized to prepare a groupof 1,4-dihydroquinoxaline-2,3-diones that otherwise could not beprepared easily. Nucleophilic substitution of the fluoro ortho to thenitro group in 6,7-difluoro-5-nitroquinoxaline-2,34-dione (1) withdifferent nucleophiles was accomplished as shown in eq 11-15. Thereaction was generally followed by ¹H NMR and/or ¹⁹F NMR spectroscopy.Replacement of the fluoro ortho to the nitro group by a nucleophileconverts the aromatic H from a doublet of doublets to a doublet. The F—Hcoupling constants are around 11.5 Hz, which confirms that the remainingF is ortho to the H. The reaction between 1 and sodium azide is almostinstantaneous after the two are mixed in DMSO. Measurement of ¹H NMRspectrum immediately after the sample was mixed showed a doublet at 7.1ppm for the aromatic H and no starting material was observed.

Similarly, the reaction of7-chloro-6-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione with sodiummethoxide gave 7-chloro-6-methoxy-5-nitroquinoxaline-2,3-dione, and withethanethiol gave 7-chloro-6-ethylthio-5-nitroquinoxaline-2,3-dione.

Thus, the invention also relates to a method for the preparation ofcompounds having the Formula:

or a tautomer thereof;

wherein

R¹ is nitro, cyano, CF₃, carboxy, or alkanoyl;

R² is alkoxy, aralkoxy, hydroxy, mercaptoalkyl, azido, or NR⁵R⁶, whereinR⁵ and R⁶ are independently hydrogen, alkyl, or aryl groups;

R³ is halo, haloalkyl, nitro, alkyl, alkoxy, azido, or cyano; and

R⁴ is hydrogen;

comprising reaction of a compound having the Formula:

or a tautomer thereof;

wherein

R¹ is nitro, cyano, CF₃, carboxy, or alkanoyl;

R² is fluoro;

R³ is halo, haloalkyl, nitro, alkyl, alkoxy, azido, or cyano; and

R⁴ is hydrogen;

with an alkoxide, aryl alkoxide, hydroxide, an alkyl mercaptide, azide,or HNR⁵R⁶, respectively, in an inert solvent, and isolating the compoundso produced. Examples of such inert solvents include dipolar aproticsolvents, such as DMF and DMSO. Where the nucleophile is an alkoxygroup, the solvent can be the corresponding alcohol. The reaction iscarried out between room temperature and 120° C., and the progressmonitored by NMR spectroscopy.

Electron withdrawing groups such as cyano, CF₃, carboxy, and alkanoyl inthe 5-position are expected to behave similarly to nitro. Any group canbe present in the 7- and 8-positions for the reaction to proceed.

Examples of compounds that can be produced according to this methodinclude 6-azido-7-fluoro-5-nitro-14,dihydroquinoxaline-2,3-dione;6-amino-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-6-methoxy-5-nitro -1,4-dihydroquinoxaline-2,3-dione;7-fluoro-6-ethoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-fluoro-6-hydroxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione;7-chloro-6-methoxy-5-nitroquinoxaline-2,3-dione; and7-chloro-6-ethylthio-5-nitroquinoxaline-2,3-dione.

Thus, the present invention is.directed to compounds having high bindingto the glycine receptor and low binding to the kainate and AMPA sites.The glycine antagonist potency in vitro can be determined using a 1Mglycine-stimulated [³H]-MK801 binding assay. This assay takes advantageof the dependence of the binding of [³H]-MK801 to the PCP receptorinside the pore of the NMDA channel on the presence of both glutamateand glycine. In the absence of glycine, but in the presence ofglutamate, [³H]-MK801 cannot bind effectively to the PCP receptorbecause the NMDA channel remains closed and access of [³H]-MK801 to thePCP receptor inside the closed channel pore is severely restted.

The assay is conducted using rat brain membrane homogenates that areenriched in NMDA receptors. The membranes are prepared as follows.Frozen rat brains (obtained from Pel-Freez, Rogers, Arkansas) arehomogenized in 15 volumes (w/v) of ice cold 0.32 M sucrose. Thehomogenate is spun at 1,000×g for ten minutes. The supernatant iscollected and spun for 20 minutes at 44,000×g. The pellet is suspendedin 15 volumes of water (relative to original brain weight). Thehomogenate is again spun at 44,000×g for twenty minutes. The pellet isresuspended in 5 volumes of water and the suspension is freeze-thawed 2times. After the final thaw cycle, the suspension is brought to 15volumes with water and spun at 44,000×g for twenty minutes. The pelletis resuspended in 5 volumes of ice-cold 10 mM HEPES, and is titrated topH 7.4 with KOH containing 0.04% Triton X-100. Membranes are incubatedwith the Triton/HEPES buffer at 37° C. for 15 minutes. The volume isthen brought to 15 with ice-cold 10 mM HEPES, pH 7.4, and spun/washedthree times with spins of 44,000×g between washes. The final pellet issuspended in three volumes of 50 mM HEPES, pH 7.4, and the proteinconcentration is determined with a standard dye-binding protein assay(Bio-Rad, Richmond, Calif.). The suspension is stored at −80° C. untilused. Only HPLC grade water is used for all buffers andsuspensions/washings. The extensive washings are necessary to remove asmuch endogenous glycine from the membrane preparation as possible.

On the day of the assay, the previously prepared membranes are thawedand 5 mM Tris/HCl buffer, pH 7.4, is added to yield a final proteinconcentration of 0.156 mg/mL. For binding assays, 0.8 mL of membranesare pipetted into polypropylene tubes followed by 0.033 mL of 15.1 μM5,7-dichlorokynurenic acid (D)CK), 0.033 ml of 30.3 μM glycine in buffer(or buffer alone), 0.033 mL of 303 μM glutamate in buffer (or forcontrols, 0.1 mL 1 mM PCP instead of DCK/gly/glu), 0.033 mL glycineantagonist in buffer (or buffer alone) and 0.1 mL buffer containing200,000 cpm [³H]-MK801. Nonspecific binding is defined as the differencein binding that occurs in the absence or presence of-PCP (finalconcentration: 100 μM). To determine the effect of 1 μM glycine on thebinding of [³H]-MK801, bound radioactivity in the presence of 10 μMglutamate alone (final concentration) is subtracted from the boundradioactivity in the presence of both 10 μM glutamate and 1 μM glycine(final concentration). A 500 nM concentration (final) of5,7-dichlorokynurenic (DCK) acid is added to all assay tubes. Thisconcentration of the glycine antagonist DCK “buffers” most of theresidual endogenous glycine that is not removed by the extensive washingsteps that are carried out during the membrane preparation procedure.The 500 nM DCK does not interfere with the stimulation of [³H]-MK801binding that is effected by the addition of 1 μM exogenous glycine.

The assays are incubated for 120 minutes at room temperature after whichtime the membrane-bound radioactivity is isolated from the freeradioactivity by vacuum filtration through Whatman glass fiber filtersthat had been pretreated with 0.3% polyethyleneimine. Filtration isaccomplished using a Brandel,48 well cell harvester. Filtered membranesare washed three times with 3 mL each of ice cold buffer. Filters aretransferred to scintillation vials and 5 mL of scintillation cocktail isadded. The vials are shaken overnight and the radioactivity is countedby liquid scintillation spectroscopy. The assays are done in triplicateand all experiments are conducted at least three times.

Inhibition dose response curves are constructed using increasingconcentrations of glycine antagonists from 5 nM to 330 μM. IC₅₀ D valuesare determined for compounds active in inhibiting 1 μMglycine-stimulated [³H]-MK801 binding by computer-assisted plotting ofthe inhibition curves and interpolation. When compounds are found toinhibit glycine-stimulated [³H]-MK801 binding, experiments are conductedto determine whether the inhibition of the glycine-stimulated [³H]-MK801binding is indeed mediated at the glycine binding site of the NMDAreceptor. In these experiments, a fixed concentration of antagonistsufficient to produce a >95% inhibition of the 1 μM glycine-stimulated[³H]-MK801 binding is incubated with the membranes without anyadditional glycine (above 1 μM) and in the presence of increasingconcentrations of additional glycine (2 μM to 1 μM). If the inhibitionof [³H]-MK801 binding by the drug in the presence of 1 μM glycine isfully reversed by adding increasing concentrations of glycine, then theinhibition of [³H]-MK801 binding is mediated by the drug acting as anantagonist at the glycine binding site of the NMDA receptor.

After constructing inhibition dose response curves and determination ofglycine reversibility, K_(i) values for the glycine antagonists arecalculated using the Cheng and Prusoff equation employing theexperimentally determined IC₅₀ values, the known concentration ofglycine in the assay (1 μM) and the known affinity of glycine for theglycine binding site of the NMDA receptor (100 nM).

The same rat brain membrane homogenates used for the 1 μMglycine-stimulated [³H]-MK801 binding assay are used for the [³H]-AMPAradioligand binding assay. On the day of the assay the frozen membranes(prepared as described above) are thawed and diluted with 30 mM Tris/HClbuffer containing 2.5 mM CaCl₂ and 100 mM KSCN, pH 7.4, to yield a finalmembrane concentration of 1.25 mg/mL membrane protein. For the bindingassay, 0.8 mL of membrane homogenate is added to polypropylene tubesfollowed by 0.033 mL drug and 0.067 mL buffer (or, for controls, by 0.1mL buffer alone) and 0.1 mL buffer containing 200,000 cpm of [³H]-AMPA.The assay is incubated for 30 minutes on ice. Bound radioactivity isseparated from free radioactivity by filtration over Whatman glass fiberfilters (pretreated with 0.3% polyethyleneimine) using a Brandel 48 wellcell harvester.

Filtered membranes are washed three times with 3 mL each of ice coldbuffer. The filters are transferred to scintillation vials and 5 mL ofscintillation cocktail is added. The vials are shaken overnight andradioactivity is counted by liquid scintillation spectroscopy.Nonspecific binding is determined by the radioactivity that remainsbound to the membranes in the presence 10 mM glutamate. Inhibition doseresponse curves are constructed by adding increasing concentrations ofdrug from 10 nM to 100 μM.

The same membrane preparation as that used for the [³H]-AMPA bindingassay can be used for the [³H]-Kainate radioligand binding assay. On theday of the assay the frozen rat brain membranes are thawed and 5 mM aTris/HCl buffer, pH 7.4, is added to yield a final concentration of 0.5mg/mL membrane protein. For the binding assay, 0.8 mL of membranehomogenate is added to polypropylene tubes followed by 0.033 mL drug and0.067 mL buffer (or, for controls, by 0.1 mL buffer alone) and 0.1 mLbuffer containing 200,000 cpm of [³H]-kainate. The assay is incubatedfor 2 hours on ice. Bound radioactivity is separated from freeradioactivity by filtration over Whatman glass fiber filters (pretreatedwith 0.3% polyethyleneimine) using a Brandel 48 well cell harvester.Filtered membranes are washed three times with 3 mL each of ice coldbuffer. The filters are transferred to scintillation vials and 5 mL ofscintillation cocktail is added. The vials are shaken overnight andradioactivity is counted by liquid scintillation spectroscopy.Nonspecific binding is determined by the radioactivity that remainsbound to the membranes in the presence 10 mM glutamate. Inhibition doseresponse curves are constructed by adding increasing concentrations ofdrug from 250 nM to 330 μM.

The anxiolytic activity of any particular compound of the presentinvention can be determined by use of any of the recognized animalmodels for anxiety. A preferred model is described-by Jones, B. J. etal., Br. J. Pharmacol. 93:985-993 (1988). This model involvesadministering the compound in question to mice that have a high basallevel of anxiety. The test is based, on the finding that such mice findit aversive when taken from a dark home environment in a dark testingroom and placed in an area that is painted white and brightly lit. Thetest box has two compartments, one white and brightly illuminated andone black and non-illuminated. The mice have access to both compartmentsvia an opening at floor level in the divider between the twocompartments. The mice are placed in the center of the brightlyilluminated area. After locating the opening to the dark area, the miceare free to pass back and forth between the two compartments. Controlmice tend to spend a larger proportion of time in the dark compartment.When given an anxiolytic agent, the mice spend more time exploring themore novel brightly lit compartment and exhibit a delayed latency tomove to the dark compartment. Moreover, the mice treated with theanxiolytic agent exhibit more behavior-in the white compartment, asmeasured by exploratory rearings and line crossings. Since the mice canhabituate to the test situation, naive mice should always be used in thetest. Five parameters can be measured: the latency to entry into thedark compartment, the time spent in each area, the number of transitionsbetween compartments, the number of lines crossed in each compartment,and the number of rears in each compartment. The administration of thecompounds of the present invention is expected to result in the micespending more time in the larger, brightly lit area of the test chamber.

In the light/dark exploration model, the anxiolytic activity of aputative agent can be identified by the increase of the numbers of linecrossings and rears in the light compartment at the expense of thenumbers of line crossings and rears in the dark compartment, incomparison with control mice.

A second preferred animal model is the rat social interaction testdescribed by Jones, B.J. et al., supra, wherein the time that two micespend in social interaction is quantified. The anxiolytic activity of aputative agent can be identified by the increase in the time that pairsof male rats spend in active social interaction (90% of the behaviorsare investigatory in nature). Both the familiarity and the light levelof the test arena can be manipulated. Undrugged rats show the highestlevel of social interaction when the test arena is familiar and is litby low light. Social interaction declines if the arena is unfamiliar tothe rats or is lit by bright light. Anxiolytic agents prevent thisdecline. The overall level of motor activity can also be measured toallow detection of drug effects specific to social behaviors.

The efficacy of the glycine and-excitatory amino acid antagonists toinhibit glutamate neurotoxicity in a rat brain cortex neuron cellculture system can be determined as follows. An excitotoxicity modelmodified after that developed by Choi (Choi, D. W., J. Neuroscience7:357 (1987)) can be used to test anti-excitotoxic efficacy of theglycine and excitatory amino acid antagonists. Fetuses from ratembryonic day 19 are removed from time-mated pregnant rats. The brainsare removed from the fetuses and the cerebral cortex is dissected. Cellsfrom the dissected cortex are dissociated by a combination of mechanicalagitation and enzymatic digestion according to the method of Landon andRobbins (Methods in Enzymology 124:412 (1986)). The dissociated cellsare passed through an 80 micron nitex screen and the viability of thecells are assessed by Trypan Blue. The cells are plated on poly-Dysinecoated plates and incubated at 37° C. in an atmosphere containing 91%02/9% CO₂. Six days later, fluoro-d-uracil is added for two days tosuppress non-neural cell growth. At culture day 12, the primary neuroncultures are exposed to 100 μM glutamate for 5 minutes with or withoutincreasing doses of glycine and excitatory amino acid antagonist orother drugs. After 5 minutes, the cultures are washed and incubated for24 hours at 37° C. Neuronal cell damage is quantitated by measuringlactate dehydrogenase (LDH) activity that is released into the culturemedium. The LDH activity is measured according to the method of Deckeret al. (Decker et al., J. Immunol. Methods 15:16 (1988)).

The anticonvulsant activity of the glycine and excitatory amino acidantagonists can be assessed in the audiogenic seizure model in DBA-2mice as follows. DBA-2 mice can be obtained from Jackson Laboratories,Bar Harbor, Maine. These mice at an age of <27 days develop a tonicseizure within 5-10 seconds and die when they are exposed to a sound of14 kHz (sinus wave) at 110 dB (Lonsdale, D., Dev. Pharmacol. Ther. 4:28(1982)). Seizure protection is defined when animals injected with drug30 minutes prior to sound exposure do not develop a seizure and do notdie during a 1 minute exposure to the sound. 21 day old DBA-2 mice areused for all experiments. Compounds are given intraperitoneally ineither saline, DMSO, or polyethyleneglycol-400. Appropriate solventcontrols are included in each experiment. Dose response curves areconstructed by giving increasing doses of drug from 1 mg/kg to 100mg/kg. Each dose group (or solvent control) consists of at least sixanimals.

The anticonvulsant efficacy of the glycine receptor antagonists can beassessed in the pentylenetetrazol (PTZ-induced seizure test as follows.Swiss/Webster mice, when injected with 50 mg/kg PTZ (i.p.) develop aminimal clonic seizure of approximately 5 seconds in length within 5-15minutes after drug injection. Anticonvulsant efficacy of aglycine/excitatory amino acid antagonist (or other) drug is defined asthe absence of a seizure when a drug is given 30 minutes prior to PIZapplication and a seizure does not develop for up to 45 minutesfollowing PTZ administration. Glycine/excitatory amino acid antagonistor other drugs are given intraperitoneally in either saline, DMSO, orpolyethyleneglycol-400. Appropriate solvent controls are included ineach experiment. Dose response curves are constructed by givingincreasing doses of drug from 1 mg/kg to 100 mg/kg. Each dose group (orsolvent control) consists of at least six animals.

The efficacy of glycinelexcitatory amino acid antagonists to protectmice from NMDA-induced death can be assessed as follows. When mice areinjected with 200 mg/kg N-methyl-D-aspartate (NMDA) i.p., the animalswill develop seizures followed by death within 5-10 minutes.Glycine/excitatory amino acid antagonists are tested for their abilityto prevent NMDA-induced death by giving the drugs i.p. 30 minutes priorto the NMDA application. Glycine/excitatory amino acid antagonist orother drugs are given intraperitoneally in either saline, DMSO, orpolyethyleneglycol-400. Appropriate solvent controls are included ineach experiment. Dose response curves are constructed by givingincreasing doses of drug from 1 mg/kg to 100 mg/kg. Each dose group (orsolvent control) consists of at least six animals.

The anticonvulsant activity of the glycine antagonists can be assessedin the MES assays in mice. Electroshock was applied to maleSwiss/Webster mice (20-30 g, Simonsen) through corneal electrodes(Swinyard, E. A. (1973) in Anticonvulsant drugs, Mercier J. Ed. PergamonPress, Oxford pp. 47-65). The seizure stimulus parameters were: 50 mA,60 Hz, rectangular pulse, width 0.8 msec, duration 200 msec. Tonic hindlimb extension observed after application of the electrical stimulus wasrecorded as occurrence of seizure. The drug was applied i.v. as anaqueous Tris (Tromethamine) solution.

A series of different evaluations can be conducted on doses of theglycine/excitatory amino acid antagonists of the invention to determinethe biological activity of the compounds both in normal gerbils and inanimals exposed to 5 minutes of bilateral carotid occlusion. See SchemeVIII.

These studies are conducted in animals who are conscious and have noother pharmacological agents administered to them. Gerbils arepreinstrumented 48-hours prior to ischemia to allow for the completeelimination of the pentobarbital anesthetic that is employed. Whentested with drugs, animals are given i.p. injections of theglycinetexcitatory amino acid antagonist or vehicle. In the case ofmultiple injections, animals are given i.p. injections 2 hours apart andthe final injection is given 30 minutes prior to the ischemic period orin the case of post treatment, the animals are given injections at 30minutes, 2 hours, 4 hours, and 6 hours post-ischemic reperfusion.

In order to assess the direct pharmacological activity or potentialactivity of the glycine/excitatory amino acid antagonists, naive gerbilsare injected with either saline or differing doses of the antagonist.The behavioral changes are assessed using a photobeam locomotor activitychamber, which is a two foot circular diameter arena with photobeamdetection. Animals are individually placed in the 2 foot diameterchambers. The chambers are housed in a cabinet that is closed and noiseis abated using both a background white noise generator and a fan.Animals are placed in these chambers in the case of the initialpharmacological evaluation for a period of 6 hours and the totalactivity during each successive hour is accumulated using the computercontrol systems.

Saline results in an initial high rate of activity, with the controlanimals showing a first hour activity level of about 1600 counts. Thislevel of control activity is typical for the gerbil under theseexperimental conditions. As the session progresses, animals decreasetheir exploratory activity and at the terminal period the activitydeclines to about 250 counts per hour. It is expected that theglycine/excitatory amino acid antagonists of the present invention willhave no significant effect on either the initial exploratory rate or theterminal rate of exploration.

In a next phase of the evaluation of the glycine/excitatory amino acidantagonists, gerbils are pretreated with varying doses of theantagonists and then exposed to a five minute period of bilateralcarotid occlusion. Following the initiation of reperfusion, animals areplaced into the circular locomotor activity testing apparatus and theactivity at the beginning of the first hour following reperfusion ismonitored for the subsequent four hours.

Control animals not exposed to ischemia and given injections of salineprior to being placed in the locomotor activity chamber show acharacteristic pattern of activity, which in the first hour of locomotoractivity is substantially higher than during all other hours andprogressively declines over the four hours to a very low value. Incontrast to the progressive decline in activity over the four hourtesting period, control animals that are exposed to five minutes ofcortical ischemia demonstrate a completely different pattern oflocomotor activity. During the first hour, there is a significantdecline in activity that is followed by a progressive increase in whichthe activity during the fourth hour is ten-fold higher than thatdemonstrated by animals not exposed to carotid occlusion. These resultsare typical and are a reliable result of the alterations caused by fiveminutes of bilateral carotid occlusion in the gerbil. a Separate groupsof gerbils are pretreated with the glycine/excitatory amino acidantagonists of the invention 30 minutes before the onset of carotidocclusion and then placed into the locomotor activity following one hourof reperfusion. It is expected that pretreatment of the gerbils with theglycine/excitatory amino acid antagonists of the invention will preventboth the post-ischemic decrease and increase in activity. Post-ischemicdecreases in activity are expected to be near zero during the first hourfollowing reperfusion. Pretreatment with the glycine/excitatory aminoacid antagonists of the invention is expected to reduce or prevent thisearly depression of behavior. In addition, the glycine/excitatory aminoacid antagonists of the invention are expected to prevent thepost-ischemic stimulation of behavior.

Subsequent to completion of the single dose pretreatment evaluations,gerbils are also evaluated with multiple injections of theglycine/excitatory amino acid antagonists of the invention. Doses areadministered i.p. at 6 hours, 4 hours, 2 hours, and 30 minutes prior tothe onset of 5 minutes of ischemia.

At 24 hours, all animals are evaluated for differences in patrollingbehavior using an 8-arm radial maze. In this procedure, animals areplaced into the center start chamber of the maze, the barrier isremoved, and the amount of time and the number of times the animal makesan error is recorded prior to completion of exploration in all 8 arms ofthe maze. An error is defined as the revisiting of an arm by an animalentering to the extent of its entire body without including its tail. Ifthe animal perseveres or fails to leave the arm for longer than fiveminutes, the session is terminated. In the control population of theanimals, the number of errors and exploration of the maze with no priorexperience (naive) is approximately 6 errors. This is an average valuefor an N of 28 gerbils. Following 5 minutes of bilateral carotidocclusion and testing at 24 hours, gerbils make an average number oferrors of 21. When animals are pretreated with the glycine/excitatoryamino acid antagonists of the invention, there is expected to be asignificant reduction in the number of errors made. There is alsoexpected to be a significant sparing of the behavioral changes that areinduced in the radial arm maze performance.

It is also expected that post treatment with the glycine/excitatoryamino acid antagonists of the invention will reduce the short termmemory impairment 24 hours post ischemic/reperfusion.

The effects of 5 minutes of bilateral carotid occlusion on neuronal celldeath in the dorsal hippocampus can be evaluated in animals 7 days afterischemia reperfusion injury. Previous studies have demonstrated thatneuronal degeneration begins to occur around 3 days following cerebralischemia. By 7 days those neurons that have been affected will undergocytolysis and have either completed degeneration or are readily apparentas dark nuclei and displaced nuclei or as cells with eosinophiliccytoplasm and pycnotic nuclei. The lesion with 5 minutes of ischemia isessentially restricted within the hippocampus to the CA1 region of thedorsal hippocampus. The intermedial lateral zone of the horn isunaffected and the dentate gyrus and/or cells in CA3 do not showpathology. Gerbils are anesthetized on day 7 following ischemia with 60mg/kg of pentobarbital. Brains are perfused transcardiac with ice-coldsaline followed by buffered paraformaldehyde (10%). Brains are removed,imbedded, and sections made. Sections are stained with hematoxylin-eosinand neuronal cell counts are determined in terms of the number ofneuronal nuclei/100 micrometers. Normal control animals (not exposed toischemia reperfusion injury) will not demonstrate any significant changein normal density nuclei within this region. Exposure to five minutes ofbilateral carotid occlusion results in a significant reduction in thenumber of nuclei present in the CA1 region. In general, this lesionresults in a patchy necrosis instead of a confluent necrosis, which isseen if 10 minutes of ischemia is employed. Pretreatment with theglycine receptor antagonists of the invention is expected to produce asignificant protection of hippocampal neuronal degeneration.

It is known that NMDA receptors are critically involved in thedevelopment of persistent pain following nerve and tissue injury. Tissueinjury, such as that caused by injecting a small amount of formalinsubcutaneously into the hindpaw of a test animal, has been shown toproduce an immediate increase of glutamate and aspartate in the spinalcord (Skilling, S. R., et al., J. Neurosci. 10:1309-1318 (1990)).Administration of NMDA receptor blockers reduces the response of spinalcord dorsal horn neurons following formalin injection (Dickenson andAydar, Neuroscience Lett. 121:263-266 (1991); Haley, J. E., et al.,Brain Res. 518:218-226 (1990)). These dorsal horn neurons are criticalin carrying the pain signal from the spinal cord to the brain and areduced response of these neurons is indicative of a reduction in painperceived by the test animal to which pain has been inflicted bysubcutaneous formalin injection.

Because of the observation that NMDA receptor antagonists can blockdorsal horn neuron response induced by subcutaneous formalin injection,NMDA receptor antagonists have potential for the treatment of chronicpain, such as, pain caused by surgery, by amputation (phantom pain), orby infliction of other wounds (wound pain). However, the use ofconventional NMDA antagonists, such as, MK801 or CGS 19755, inpreventing or treating chronic pain is severely limited by the adversePCP-like behavioral side effects that are caused by these drugs. It isexpected that the glycine receptor antagonists that are the subject ofthis invention will be highly effective in preventing chronic pain inmice induced by injecting formalin subcutaneously into the hindpaw ofthe animals. Because the glycine/excitatory amino acid antagonists ofthis invention are expected to be free of PCP-like side effects, thesedrugs are highly useful in preventing or treating chronic pain withoutcausing PCP-like adverse behavioral side effects.

The effects of the glycine receptor antagonists of the present inventionon chronic pain can be evaluated as follows. Male Swiss/Webster miceweighing 25-35 grams are housed five to a cage with free access to foodand water and are maintained on a 12 hour light cycle (light onset at0800h). The glycine receptor antagonist is dissolved in DMSO at aconcentration of 140 and 5-40 mg/mL, respectively. DMSO is used asvehicle control. All drugs=are injected intraperitoneally (1 μl/g). Theformalin test is performed -as described (Dubuisson and Dennis, Pain4:H161-174 (1977)). Mice are observed in a plexiglass cylinder, 25 cm indiameter and 30 cm in height. The plantar surface of one hindpaw isinjected subcutaneously with 20 μl of 5% formalin. The degree of pain isdetermined by measuring the amount of time the animal spends licking theformalin-injected paw during the following time intervals: 0-5′ (earlyphase); 5′-10′, 10′-15′ and 15′-50′ (late phase). To test whether theglycine/excitatory amino acid antagonists prevent chronic pain in thetest animals, vehicle (DMSO) or drugs dissolved in vehicle at doses of 1mg/kg to 40 mg/kg are injected intraperitoneally 30 minutes prior to theformalin injection. For each dose of drug or vehicle control at leastsix animals are used.

Compared to vehicle control, it is expected that the intraperitonealinjection of the glycine receptor antagonists 30 minutes prior toformalin injection into the hindpaw will significantly inhibitformalin-induced chronic pain in a dose-dependent manner as determinedby the reduction of the time the mouse spends licking the formalininjected hindpaw, caused by increasing doses of glycine/excitatory aminoacid antagonist.

It is well known to use opiates, e.g., morphine, in the medical field toalleviate pain. (As used herein, the term “opiates” is intended to meanany preparation or derivative of opium, especially the alkaloidsnaturally contained therein, of which there are about twenty, e.g.,morphine, noscapine, codeine, papaverine, and thebaine, and their-derivatives.) Unfortunately, with continued use, the body builds up atolerance for the opiate, and, thus, for continued relief, the patientmust be subjected to progressively larger doses. This, in itself, can bedetrimental to the patient's health. Furthermore, a time can come whenthe tolerance is substantially complete and the pain killing propertiesof the drug are no longer effective. Additionally, administration ofhigher doses of morphine may lead to respiratory depression, causing thepatient to stop breathing. Recent studies have suggested a modulatoryrole for the NMDA receptor in morphine tolerance. It has now been foundthat administration of quinoxaline diones can inhibit opiate toleranceby blocking the glycine co-agonist site associated with the NMDAreceptor.

Compositions within the scope of this invention include all compositionswherein the compounds of the present invention are contained in anamount effective to achieve its intended purpose. While individual needsvary, determination of optimal ranges of effective amounts of eachcomponent is within the skill of the art. Typically, the compounds maybe administered to mammals, e.g., humans, orally at a dose of 0.0025 to50 mg/kg, or an equivalent amount of the pharmaceutically acceptablesalt thereof, per day of the body weight of the mammal being treated foranxiety disorders, e.g., generalized anxiety disorder, phobic disorders,obsessional compulsive disorder, panic disorder, and post traumaticstress disorders. Preferably, about 0.01 to about 10 mg/kg is orallyadministered to treat or prevent such disorders. For intramuscularinjection, the dose is generally about one-half of the oral dose. Forexample, for treatment or prevention of anxiety, a suitableintramuscular dose would be about 0.0025 to about 15 mg/kg, and mostpreferably, from about 0.01 to about 10 mg/kg.

In the method of treatment or prevention of neuronal loss in ischemia,brain and spinal cord trauma, hypoxia, hypoglycemia, and surgery, aswell as for the treatment of Alzheimer's disease, amyotrophic lateralsclerosis, Huntington's disease, and Down's Syndrome, or in a method oftreating a disease in which the pathophysiology of the disorder involveshyperactivity of the excitatory amino acids or NMDA receptor-ion channelrelated neurotoxicity or psychosis, the pharmaceutical compositions ofthe invention can comprise the compounds of the present invention at aunit dose level of about 0.01 to about 50 mg/kg of body weight, or anequivalent amount of the pharmaceutically acceptable salt thereof, on aregimen of 14 times per day. When used to treat chronic pain or toinduce anesthesia, the compounds of the invention may be administered ata unit dosage level of from about 0.01 to about 50 mg/kg of body weight,or an equivalent amount of a pharmaceutically acceptable salt thereof,on a regimen of 14 times per day. Of course, it is understood that theexact treatment level will depend upon the case history of the animal,e.g., human being, that is treated. The precise treatment level can bedetermined by one of ordinary skill in the art without undueexperimentation.

The unit oral dose may comprise from about 0.01 to about 50 mg,preferably about 0.1 to about 10 mg of the compound. The unit dose maybe administered one or more times daily as one or more tablets eachcontaining from about 0.1 to about 10, conveniently about 0.25 to 50 mg,of the compound or its solvates.

In addition to administering the compound as a raw chemical, thecompounds of the invention can be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries thatfacilitate processing of the compounds into preparations that can beused pharmaceutically. Preferably, the preparations, particularly thosepreparations that can be administered orally and that can be used forthe preferred type of administration, such as tablets, dragees, andcapsules, and preparations that can be administered rectally, such assuppositories, as well as suitable solutions for administration byinjection or orally, contain from about 0.01 to 99 percent, preferablyfrom about 0.25 to 75 percent of active compound(s), together with theexcipient.

Also included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. Basic salts are formed by mixing a solution of theparticular 1,4-dihydroquinoxaline-2,3-dione of the present inventionwith a solution of a pharmaceutically acceptable non-toxic base, suchas, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodiumcarbonate, or an amino compound, such as, choline hydroxide, Tris,bis-Tris, N-methylglucamine, arginine, and the like. See, U.S.application Ser. No. 08/148,268, supra.

The pharmaceutical compositions of the invention can be administered toany animal that may experience the beneficial effects of the compoundsof the invention. Foremost among such animals are humans, although theinvention is not intended to be so limited.

The pharmaceutical compositions of the present invention can beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, or ocular routes.Alternatively, or concurrently, administration may be by the oral route.The dosage administered will be dependent upon the age, health, andweight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired.

When the compositions of the invention are administered ocularly, onemay achieve either local or systemic administration. For example, thecompositions of the present invention may be administered in the form ofeye drops that are substantially isotonic with tear fluid to achievesystemic administration. Preferably, such compositions will alsocomprise a permeation-enhancing agent, which aids the systemicabsorption of the compounds of the present invention. See, U.S. Pat. No.5,182,258. Alternatively, the compositions of the invention may beadministered ocularly to treat or prevent optic nerve degeneration. Inthis embodiment, the compounds of the present invention are administeredin the form of eye drops, as disclosed above, or may be injected intothe vicinity of the optic nerve. In the alternative, thin ocularimplants may be employed that slowly release the compounds of thepresent invention.

In addition to the pharmacologically active compounds, the newpharmaceutical preparations can contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries thatfacilitate processing of the active compounds into preparations that canbe used pharmaceutically.

The pharmaceutical preparations of the present invention aremanufactured in a manner that is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers, such as, saccharides,for example, lactose or sucrose, mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example, tricalciumphosphate or calcium hydrogen phosphate, as well as binders, such as,starch paste, using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone. If desired, disintegrating agents can be added,such as, the above-mentioned starches and also carboxymethyl-starch,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as, sodium alginate. Auxiliaries are, above all,flow-regulating agents and lubricants, for example, silica, talc,stearic acid or salts thereof, such as, magnesium stearate or calciumsteamate, and/or polyethylene glycol. Dragee cores are provided withsuitable coatings that, if desired, are resistant to gastric juices. Forthis purpose, concentrated saccharide solutions can be used, which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. In order to produce coatings resistant togastric juices, solutions of suitable cellulose preparations, such as,acetyl-cellulose phthalate or hydroxypropylmethyl-cellulose phthalate,are used. Dye stuffs or pigments can be added to the tablets or drageecoatings, for example, for identification or in order to characterizecombinations of active compound doses.

Other pharmaceutical preparations that can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as, glycerol or sorbitol. Thepush-fit capsules can contain the active compounds in the form ofgranules that may be mixed with. fillers, such as, lactose, binders,such as, starches, and/or lubricants, such as, talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds are preferably dissolved or suspended in suitable liquids,such as, fatty oils or liquid paraffin. In addition, stabilizers may beadded.

Possible pharmaceutical preparations that can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules, which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400 (thecompounds are soluble in PEG-400). Aqueous injection suspensions maycontain substances that increase the viscosity of the suspension, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers.

The characterization of glycine binding sites in vitro has beendifficult because of the lack of selective drug ligands. Thus, theglycine ligands of the present invention can be used to characterize theglycine binding site. The particularly preferred compounds that an beused for this purpose are isotopically radiolabel led derivatives, e.g.,where one or more of the atoms are replaced with ³H, ¹¹C, ¹⁴C, ¹⁵N, or¹⁸F. Examples of preferred photoaffinity ligands are ³H or¹⁸F-substituted 6-azido-5,7-difluoro-1,4-dihydroquinoxaline-2,3-dioneand ³H-substituted6-azido-5,7-dichloro-1,4-dihydroquinoxaline-2,3-dione.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and obvious to those skilled in the artare within the spirit and scope of the invention.

EXAMPLES Example 1

Preparation of 6,7-Difluoro-5-nitro-1,4-dihydro-2,3-quinoxalinedione

Preparation of 6,7-Difluoro-1,4-dihydro-2,3-quinoxalinedione

Method 1.

4,5-Difluoro-1,2-diaminobenzene.

4,5-Difluoro-1,2-diaminobenzene was prepared using an adaptation of themethod of Tsuji et al., Crsuji, Y. et al., J. Org. Chem. 55:580 (1990)).Zn powder (942 mg, 14.4 mmol), CaCl₂ (94.4 mg), H₂O (1.0 mL) and 4.0 mLEtOH were combined and brought to reflux as described for4-fluoro-1,2-diaminobenzene (see Example 11) and to this mixture wasadded slowly dropwise a solution of 4,5-difluoro-2-nitroaniline (200 mg,1.15 mmol) in 2 mL ETOH. Analysis and workup were as described for4-fluoro-1,2-diaminobenzene (Example 11) except that the reaction wasdissolved in 5 mL H₂O and the solution extracted with 3×10 mL Et₂O. Theorganic layers were combined and treated with activated charcoal, dried(MgSO₄), and filtered through a pad of Celite. The solvent wasevaporated at reduced pressure to yield 111.5 mg (67.3%) of a browncrystalline solid. ¹H NMR (CDCl₃) δ 3.34 (br s, 4H, NH₂, 6.53 (t, 2H,ArH).

6,7-Difluoro-1,4-dihydro-2,3-quinoxalinedione.

The title compound (Sarges, R. et al., J. Med. Chem. 33.2240 (1990)) wasprepared using an adaptation of the method of Cheeseman. (Cheeseman, G.W. H. J. Cam. Soc. 1171 (1962)). A mixture of diethyl oxalate (1.11 g,7.63 mmol and 4,5-difluoro-1,2-diaminobenzene (110 mg, 0.763 mmol) washeated to reflux under N₂ for 2 h. The reaction was allowed to cool toroom temperature and the solid was collected by vacuum filtration,rinsed with hexanes, and air dried. This gray brown solid wasrecrystallized from 20 mL of EtOH and the brown-white crystals collectedby vacuum filtration and the crystals further dried under vacuum (0.5torr, 25° C.) to yield 45.3 mg (30.0%); mp >360° C. (lit. >310° C.); ¹HNMR (d₆-acetone) δ 7.19 (t, 2H, ArH, J_(H-F)=9.3), 10.9 (br s, 2H, NH).

Method 2.

To a solution of 2.0 g(11.5 mmol)4,5-difluoro-2-nitroaniline (Aldrich,used as received) in EtOH (20 mL) was added 100 mg of 10% Pd/C. Thesuspension was shaken under H₂ (40-20 psi) for 3 h. The catalyst wasremoved by filtration and washed with EtOH (2×15 mL). The EtOH solutionwas rota-evaporated to dryness. To the residual black solid was addedoxalic acid dihydrate (1.74 g, 13.8 mmol) and 2 N aq HCl (18 mL). Themixture was heated at 125° C. with stirring for 3 h, then cooled to 25°C. The black precipitate was collected by vacuum filtration, and washedwith water (5×5 mL). The wet product was dissolved in 0.2 N aq NaOH (100mL) with stirring, then filtered. The clear, slightly orange-yellow,filtrate was acidified by the addition of 2 N aq HCl with stirring to pH˜4. The off-white precipitate was collected by vacuum filtration, washedwith water, and dried under 1 mm Hg at 40° C. to give 1.83 g (89%) ofthe title compound as a cream colored powder. Mp >360° C. IR (KBr) 3454,3120, 1708, 1530, 1400, 1298 cm-⁻¹. ¹H NMR (DMSO-d₆) 11.939 (s, 2H),7.054 (m, 2H).

To a suspension of 6,7-difluoro-1,4-dihydro-2,3-quinoxalinedione (837mg, 4.23 mmol) in trifluoroacetic acid (30 mL) was added KNO₃ (512 mg,5.07 mmol). The mixture was stirred at 55° C. for 20 h. At the end ofthis time, 256 mg (2.50 mmol) of KNO₃ was added and the reaction mixturewas stirred at 55° C. for 20 h, then another 256 mg (2.50 mmol) of KNO₃was added and the mixture was stirred at 55° C. for 20 h. The reactionmixture was then rota-evaporated to dryness. Icecold water (about 15 mL)was added to the residual solid. The solid was collected by vacuumfiltration, washed with ice-cold water (5×5 mL), and dried at 40° C.under 1 mm Hg for 14 h, giving 700 mg (68%) of the title compound as ayellow powder. Mp 288-90° C. (dec.). IR (KBr) 3424, 3226, 1752, 1717,1554, 1356, 1304 cm⁻¹. ¹H NMR (DMSO-d₆): 12.249 (s, 1H), 11.864 (bs,1H), 7.330 (dd, 1H, J=10.5, 7.8 Hz). Analysis for C₈H₃F₂N₃O₄, calcd: C,39.50, H, 1.24, N, 17.29; found: C, 39.42, H, 1.26, N, 17.08.

Example 2

Preparation of 5,6,7-Trifluoro-1,4-dihydro-2,3-quinoxalinedione

2,3,4-Trifluoroacetanilide.

To a pink solution of 2,3,4-trifluoroaniline (1.04 g, 9.45 mmol) inchloroform (12 mL) was added acetic anhydride (1.63 g, 16.0 mmol),giving a pale pink solution that was stirred overnight under nitrogen.The chloroform was removed in vacuo to give 1.35 g (99%) of theacetanilide as a white solid: ¹H NMR (CDCl₃), 2.28 (s, 3H), 6.95 (m,1H), 7.31 (m, 1H), 7.97 (m, 1H).

3,4,5-Trifluoro-1,2-phenylenediamine.

To 2,3,4-trifluoroacetanilide (1.35 g, 7.09 mmol) is added concentratedsulfuric acid (8 mL). While the flask is in an ice bath, KNO₃ is slowlyadded, giving a tan mixture, that is stirred overnight. The reactionmixture, now dark red, is then added to ice water (45 mL), instantlygiving an orange precipitate. The volatile compound, presumably2,3,4-trifluoro-6-nitroaniline, is dissolved in ethyl acetate (18 mL)and ethyl alcohol (12 mL). To the orange solution is added stannouschloride dihydrate (7.6 g, 34 mmol). The resulting mixture is stirredand brought to reflux under N₂ for 4 h. The mixture is added to icewater (40 mL) and basified with 2N NaOH (40 mL). It is extracted withethyl acetate (3×20 mL) and the combined extracts are washed with water(20 mL) and brine (20 mL). This dark red solution is dried (MgSO₄) andevaporated to give 285 mg (25%) of the diamine as a dark red solid. ¹HNMR (CDCl₃), 3.22 (br s, 2H), 3.46 (br s, 2H), 6.32 (m, 1H).

5,6,7-Trifluoro-1,4-dihydro-2,3-quinoxalinedione.

To a brown solution of 3,4,5-trifluoro-1,2-phenylenediamine (285 mg,1.75 mmol) in aqueous 2N HCl (10 mL) is added oxalic acid (221 mg, 1.75mmol). The brown mixture is brought to reflux and stirred under N₂overnight. The mixture is filtered to yield 139 mg (37%) of crude titlecompound. An analytical sample is prepared by dissolving 42 mg of thisbrown powder in 2.5 mL boiling ethanol. Upon cooling, brown, rod-like,crystals are formed, which are filtered and dried in vacuo to yield 15mg (36%) of pure title compound: ¹H NMR (DMSO-d₆), 6.91 (m, 1H), 12.02(s, 1), 12.19 (s, 1H); analysis calculated for C₈H₃F₃N₂O₂: C, 44.46; H,1.40; N, 12.96. Found: C, 44.42; H, 1.16; N, 12.76.

Example 3

Preparation of 5-Nitro-6,7,8-trifluoro-4,4-dihydro-2,3-quinoxalinedione

To 5,6,7-trifluoro-1,4-dihydro-2,3-quinoxalinedione (93 mg, 0.43 mmol)is added concentrated sulfuric acid (0.5 mL). While the flask is in anice bath, KNO₃ is slowly added, giving a brown mixture, which is stirredovernight. The reaction mixture, now dark red, is then added to icewater (5 mL), instantly giving an orange precipitate, which is collectedby centrifugation. The powder is crystallized from ETOH (5 mL) and driedin vacuo to yield 24 mg (20%) of pale orange microcrystals. ¹H NMR(DMSO-d₆), 11.9 (br s, 1H), 12.6 (fr s, 1H).

Example 4

Preparation of 6-Chloro-5,7-difluoro-1,4-dihydroquinoxaline-2,3-dione

3-Chloro-2,4-difluoro(trifluoroacetamido)benzene.

To a solution of 10.5 g (64.3 mmol) of 3-chloro-2,4-difluoroaniline in25 mL of dioxane kept in an ice-bath was added dropwise 10 mL (14.8 g,70.4 mmol) of trifluoroacetic anhydride. The solution was stirred atroom temperature for 20 h. It was then added to 150 mL of ice-water andthe mixture was stirred for 1 h. It was filtered, washed with water, anddried to leave an almost colorless solid 16.1 g (96%); mp 73-74° C.; ¹HNMR (CDCl₃), 7.068 (m, 1), 7.976 (mb, 1), 8.146 (m, 1).

3-Chloro-2,4-difluoro-6-nitro-(trifluoroacetamido)benzene.

To a solution of 15.1 g (58.1 mmol) of3-chloro-2,4-difluoro-(trifluoroacetamido)benzene in 80 mL of H₂SO₄ keptin an ice-bath was added dropwise 10 mL of HNO₃. Solid precipitate wasobserved during addition of HNO₃. The mixture was stirred in an ice-bathfor 4 h, then added to 600 mL of ice-water. The precipitate wasfiltered, washed with water, and dried to leave an almost colorlesssolid (16.8 g, 95%); mp 124-125° C.; ¹H NMR (CDCl₃), 7.69 .(dd, 1),8.936 (mb, 1).

3-Chloro-2,4-difluoro-6-nitroaniline.

A solution of 6.35 g (20.8 mmol) of3-chloro-2,4-difluoro-6-nitro-(trifluoroacetamido)benzene in 60 mL of 7%K₂CO₃ methanol/water (3:2) was stirred at 25° C. for 4 h. The solutionwas evaporated to remove the methanol. Solid was observed in theresidue. It was filtered, washed with water, and dried to leave 301 mgof a crystalline yellow solid; mp 96-97° C.; ¹H NMR (CDCl₃), 6.07 (mb,2), 7.815 (dd, 1, J=1.92, 9.13). More solid was observed after themother aqueous solution was allowed to stand at room temperatureovernight. It was filtered, washed with water, and dried to leave ayellow solid (1.01 g). More solid was crystallized from the mothersolution. It was collected three more times to provide 2.48 g of ayellow solid. ¹H NMR is identical with above, total yield 3.80 g (87%).

4-Chloro-3,5-difluoro-1,2-phenylenediamine.

A solution of 348 mg (1.67 mmol) of 3-chloro-2,4-difluoro-6-nitroanilineand 1.57 g (8.28 mmol) of SnCl₂ in 8 mL of ethanol was heated at 70° C.for 2 h. The solution was evaporated to remove the ethanol. The residuewas treated with 2N NaOH to pH=13. White precipitate was observed. Themixture was extracted with CHCl₃ (3×10 mL). The extract was dried(MgSO₄) and evaporated to leave a red solid (285 mg, 95%); mp 77-78° C;¹H NMR (CDCl₃), 3.156 (b, 2), 3.704 (b, 2), 6.352 (dd, 1, J=1.86, 9.95).

6-Chloro-5,7-difluoro-1,4-dihydroquinoxaline-2,3-dione. A mixture of 230mg (1.29 mmol) of 4-chloro-3,5-difluoro-1,2-phenylenediamine and 125 mg(1.38 mmol) of oxalic acid in 4 mL of 2N HCl was refluxed for 3 h andcooled to room temperature. The mixture was filtered, washed with water,and dried to leave a brown solid (245 mg, 82%); mp>250° C.; ¹H NMR(DMSO-d₆), 6.921 (d, 1, J=9.61), 12.143 (s, 1), 12.168 (s, 1). MS, 232(M⁺; 100), 204 (80), 176 (40), 149 (70), 171 (80). HRMS calcd for C₈H₃³⁵ClF₂N₂O₂, 231.9848, found 231.9851.

Example 5

Preparation of7-Chloro-6,8-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione

To a solution of 120 mg (5.16 mmol) of6-chloro-5,7-difluoro-1,4-dihydroquinoxaline-2,3-dione in 1 mL of H₂S₄(97%) kept in an ice bath was added portionwise 60 mg (0.59 mmol) ofKNO₃. The solution was stirred at room temperature for 14 h and 60 mg ofKNO₃ was added. It was stirred at room temperature for 24 h, thendiluted with ice-water (4 mL), filtered, washed with water, and dried toleave a yellow solid (94 mg, 65%). The solid was purified by NaOH/HClprecipitation to leave 63 mg of yellow solid; mp>250° C.; ¹H NMR(DMSO-d₆), 12.11 (mb, 1), 12.503 (s, 1).

Example 6

Preparation of 7-Fluoro-5-nitro-1,4-dihydro-2,3-quinoxalinedione

4-Fluoro-2,6-dinitroaniline.

A solution of 4-fluoro-2,6-dinitro(trifluoroacetamido)benzene (297 mg,1.00 mmol) in 10% K₂CO₃ (10 mL) was refluxed for 1 h, then cooled toroom temperature to give yellow crystals. It was filtered and washedwith cold water (2×1 mL), affording 105 mg (52%) of the title compound.¹H NMR (DMSO-d₆): δ 8.254 (s, 2H), 8.460 (d, 2H, J=8.4).

1,2-Diamino-4-fluoronitrobenzene.

A solution of 4-fluoro-2,6-dinitroaniline (125 mg, 0.62 mmole) infreshly prepared 6% (NH₄)₂S (5 mL) and EtOH (5 mL) was refluxed for 30min, diluted with water (10 mL,) and kept at 4° C. for several hours.The precipitate was collected and washed with cold water (2×1 mL),affording 53 mg (50%) of 1,2-diamino-4-fluoro-6-nitrobenzene as redcrystals. ¹H NMR (CDCl₃: δ 3.619 (s, 2H), 5.724 (s, 2H), 6.732 (dd, 1H,J₁=2.4 Hz, J₂=8.4 Hz), 7.403 (dd, 1H, J₁=2.4 Hz, J₂=8.4 Hz).

7-Fluoro-5-nitro-1,4-dihydro-2,3-quinoxalinedione.

A mixture of 1,2-diamino-4-fluoro-6-nitrobenzene (80 mg, 0.46 mmole) andoxalic acid dihydrate (70 mg, 0.56 mmole, used as received) in 4N HCl (4mL) was refluxed at 120-5° C. for 3 h, then cooled to room temperature.The mixture was centrifuged and the supernatant was removed. The yellowsolid was washed with cold water (2×2 mL), collected by filtration, anddried in vacuo for 2 h, affording 45 mg (42%) of crude7-fluoro-5-nitro-1,4-dihydro-2,3-quinoxalinedione as a yellow powder.The crude product was taken up in 1N NaOH (1 mL) and filtered. Thefiltrate was acidified to pH=3, affording 35 mg (33%) of7-fluoro-5-nitro-1,4-dihydro-2,3-quinoxalinedione; mp: 333-335° C.(dec.); IR (KBr, cm⁻¹): 3427, 3328, 3104, 3072, 1716, 1545. ¹H NMR(DMSO-d₆): δ 12.418 (s, 1H), 11.149 (s, 1H), 7.819 (dd, J₁=2.4 Hz,J₂=39.0 Hz, 1H), 7.297 (dd, J₁=2.4 Hz, J₂=9.0 Hz, 1H); HRMS: calcd forC₈H₄FN₃O₄ (M⁺) m/z: 225.0185; found: 225.0188.

Example 7

Preparation of6-Chloro-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione

2-Chloro-4-fluoro-5-nitrotoluene.

To a stirred solution of 2-chloro-4-fluorotoluene (2.000 g, 1.383 mmol,Aldrich, used as received) in conc. H₂SO₄ (15.0 mL) at ° C., KNO₃ (1.400g, 1.385 mmol) was added in one lot. The resulting pale yellow solutionwas allowed to warm to 28° C. and stirred overnight at 28° C. It wasthen poured into ice (100 g) and extracted with ethyl acetate (2×100mL). Ethyl acetate was dried over anhydrous Na₂SO₄, removed undervacuum, and the resulting oil was dried further under vacuum to afford2.085 g (80%) of title compound as an oil, which was used as such forthe next reaction; ¹H NMR (CDCl₃): δ 2.422 (s, 3H), 7.325 (d, 1H, J=10.2Hz), 7.973 (d, 1H, J₁=5.2 Hz).

N-(5′-Chloro-4′-methyl-2′-nitrophenyl)glycine sodium salt.

To a stirred solution of 2-chloro-4-fluoro-5-nitrotoluene (2.080 g,10.97 mmol, as prepared above) in DMF (11.0 mL) at 70° C., was addeddropwise, a solution of sodium glycinate (1.065 g, 10.97 mmol, Aldrich,used as received) in water (11.0 mL). The resulting suspension wasstirred overnight at 70° C., cooled to room temperature, and theresulting red solid was filtered, washed with acetone (35 mL), and driedunder vacuum to give 1.005 g (37%) of the pure (¹H NMR) title compoundas a red powder, ¹H NMR (DMSO-d₆): δ 2.184 (s, 3H), 3.396 (d, 2H, J=4.2Hz), 6.852 (s, 1H), 7.991 (s, 1H), 8.671 (s, 1H).

6-Chloro-3,4-dihydro-7-methylquinoxaline-2(1H)-one.

A solution of N-(5′-chloro-4′-methyl-2′-nitrophenyl)glycine (1.000 g,4.088 mmol, as prepared above) and tin (II) chloride dihydrate (2.767 g,12.26 mmol, Aldrich, used as received) in ethanol (20.0 mL) was refluxedfor 30 min. It was then cooled to room temperature ad the precipitatedsolid was filtered, washed with ethanol (4.0 mL), and dried under vacuumto yield 0.251 g (31%) of the title compound as a yellow powder; ¹H NMR(DMSOd₆): δ 2.093 (s, 3H), 3.657 (d, 2H, J=1.5 Hz), 5.984 (s, 1H), 6.583(s, 1H), 6.632 (s, 1H), 10.253 (s, 1H).

6-Chloro-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione.

To a stirred suspension of6-chloro-3,4-dihydro-7-methylquinoxaline-2(1H)one (0.150 g, 0.763 mmol,as prepared above) in CF₃COOH (1.6 mL), excess fuming HNO₃ (0.40 mL) wasadded and the resulting red solution was stirred overnight at 28° C. Thesolvent was removed under vacuum and the residue was diluted with water(3.0 mL). The precipitated solid was filtered and dried under vacuum toyield 0.151 g (77%) of pure (purity by HPLC −100%) title compound as alight yellow powder; m.p. darkens at 350° C.; ¹H NMR (DMSO-d₆): δ 2.311(s, 3H), 7.149 (s, 1H), 12.107 (s, 1H) 12.193 (s, 1H); Elementalanalysis for CH₆ClN₃O₄: calcd C, 42.29%; H, 2.37%; N, 16.44%, found C,42.46%; H, 2.10%; N, 16.33%.

Example 8

Preparation of7-Chloro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione

2-Chloro-5-fluoro-4-nitrotoluene.

To a stirred solution of 2-chloro-5-fluorotoluene (0.500 g, 3.46 mmol,Lancaster, used as received) in conc. H₂SO₄ (5.0 mL) at 0° C., KNO₃(0.350 g, 3.46 mmol) was added in one lot. The resulting pale yellowsolution was allowed to warm to 28° C. and stirred overnight at 28° C.It was then poured into ice (50 g) and extracted with ether (2×50 mL).The ether was dried over anhydrous Na₂SO₄, removed under vacuum, and theresulting oil was dried further under vacuum to afford 0.616 g (94%) ofthe title compound as an oil, which was used as such for the nextreaction; ¹H NMR (CDCl₃): δ 2.459 (s, 3H), 7.193 (d, 1H, J₁=11.1 Hz),8.083 (d, 1H, J₁=6.6 Hz).

N-(4′-Chloro-5′-methyl-2′-nitrophenyl)glycine sodium salt. To a stirredsolution of 2-chloro-5-fluoro-4-nitrotoluene (0.605 g, 3.19 mmol, asprepared above) in DMF (3.0 mL). at 70° C., was added dropwise, asolution of sodium glycinate (0.310 g, 3.19 mmol, Aldrich, used asreceived) in water (3.0 mL). The resulting suspension was stirredovernight at 70° C. The suspension was cooled to room temperature andthe resulting red solid was filtered, washed with chloroform (10 mL) anddried under vacuum to give 0.360 g (46%) pure (¹H NMR) title compound asa red powder; ¹H NMR (DMSO-d₆): δ 2.276 (s, 3H), 3.431 (d, 2H, J=4.2Hz), 6.848 (s, 1H), 7.963 (s, 1H), 8.773 (s, 1H).

7-Chloro-3,4-dihydro-6-methylquinoxaline-2(1H)-one. A solution ofN-(4′-chloro-5′-methyl-2′-nitrophenyl)glycine sodium salt (0.300 g, 1.23mmol, as prepared above) and tin (11) chloride dihydrate (0.830 g, 3.68mmol, Aldrich, used as received) in ethanol (4.0 mL) was refluxed for 30min. It was then cooled to room temperature and the precipitated solidwas filtered, washed with ethanol (1.0 mL) and dried under vacuum toyield 0.160 g (66%) of the title compound as a yellow powder; ¹H NMR(DMSO-d₆): δ 2.099 (s, 3H), 3.655 (s, 2H), 6.037 (s, 1H), 6.538 (s, 1H),6.685 (s, 1H), 10.241 (s, 1H).

7-Chloro-6-methyl-5-nitroquinoxaline-2(1H),3(4B)-dione.

To a stirred suspension of7-chloro-3,4-dihydro-6-methylquinoxaline-2(1H)-one (0.100 g, 0.509 mmol,as prepared above) in CH₃COOH (3.0 mL), excess fuming HNO₃ (0.30 mL) wasadded and the resulting red solution was stirred overnight at 28° C. Thesolvent was removed under vacuum and the residue diluted with water (4.0mL). The precipitated solid was filtered and dried under vacuum to yield0.067 g (52%) of pure (purity by HPLC: 100%) title compound as a lightyellow powder; m.p.-darkens at 340° C.; ¹H NMR (DMSOd₆) δ 2.184 (s, 3H),7.263 (s, 1H), 11.948 (s, 1H), 12.144 (s, 1H); Elemental analysis forC₉H₆CN₃O₄.H₂O calcd C, 40.85%; H, 2.28%; N, 15.87%; found C, 40.63%; H,2.05%; N, 15.75%.

Example 9

Preparation of 6-Bromo-7-chloro-5-nitro-1,4-dihydroquinoxaline-2,3-dione

2,5-Dichloro-4-nitrobromobenzene:

A solution of 2-bromo-1,4-dichlorobenzene (1.000 g, 4.443 mmol, Aldrich,used as received) in fuming HNO₃ (7.0 mL) was stirred at 50° C. for 1.5h and poured into ice (80 g). Yellowish white solid was filtered, washedwith water (10 mL), and dried under vacuum to obtain 1.14 g (95%) ofpure (CH NMR) title compound as a yellowish white powder; m.p. 50-53° C.(lit m.p. 57-58° C.; Fox, D. L. and Turner, E. E., J. Chem. Soc. 1859(1930)); ¹H NMR (CDCl₃): δ 7.863 (s, 1H), 8.030 (s, 1H). This materialwas used as such for the next reaction.

N-(5′-Bromo-4′-chloro-2′-nitrophenyl)glycine sodium salt:

To a stirred solution of 2,5-dichloro-4-nitrobromobenzene (1.000 g,3.691 mmol, as prepared above) in DMF (10.0 mL) at 65° C., was added,dropwise, a solution of NaHCO₃ (0.316 g, 3.76 mmol) and glycine (0.280g, 3.73 mmol, Aldrich, used as received) in water (3.8 mL). Theresulting suspension was stirred at 65° C. for 65 h. The bright orangesuspension was then cooled to room temperature, filtered, washed withwater (1.0 mL), and dried under vacuum to afford 0.284 g (98%, based onrecovered starting material) of the pure (¹H NMR) title compound as anorange powder, m.p. 264-265° C. (decomposed); ¹H NMR (DMSO-d₆): δ 3.451(d, 2H, J=3.9 Hz), 7.199 (s, 1H), 8.130 (s, 1H), 8.793 (t, 1H, J=3.6Hz). It was used as such for the next reaction.

6-Bromo-7-chloro-3,4-dihydroquinoxaline-2(1H)-one:

A solution of N-(5′-bromo-4′-chloro-2′-nitrophenyl)glycine sodium salt(0.255 g, 0.824 mmol, as prepared above) and tin (II) chloride dihydrate(0.560 g, 2.48 mmol, Aldrich, used as received) in ethanol (6 mL) wasrefluxed for 3 h. It was then cooled to room temperature and allowed tostand overnight at room temperature. The white solid was filtered anddried to yield 0.038 g (18%) of the pure (¹H NMR) title compound aswhite flakes; m.p. 230-232° C.; ¹H NMR (DMSO-d₆): δ 3.732 (s, 2H), 6.371(s, 1H), 6.830 (s, 1H), 6.912 (s, 1H), 10.437 (s, 1H). Extraction of thefiltrate with ethyl acetate (30 mL) gave 0.136 g (68%) more product fora combined yield of 86%. The product was used as such for the nextreaction.

6-Bromo-7-chloro-5-nitroquinoxaline-2(1H)-one: To a stirred suspensionof 6-bromo-7-chloro-3,4-dihydroquinoxaline-2(1H)-one (0.06 g, 0.23 mmol,as obtained above) in CF₃COOH (0.5 mL) was added fuming HNO₃ (0.02 mL,0.46 mmol) and the resulting reddish yellow suspension was stirred atroom temperature overnight The cream colored suspension so obtained waspoured into ice (2.5 mL) and the precipitated solid was filtered, washedwith water (1.0 mL), and dried under vacuum to afford 0.049 g (70%) ofthe title compound as a cream colored powder. ¹H NMR (DMSO-d₆): δ 7.577(s, 1H), 8.251 (s, 1H), 12.879 (s, 1H). The crude product was used assuch for the final reaction.

6-Bromo-7-chloro-5-nitro-1,4-dihydroquinoxaline-2,3 one:

To a stirred solution of 6-bromo-7-chloro-5-nitroquinoxaline-2(1H)-one(0.025 g, 0.082 mmol, as prepared above) in conc. H₂SO₄ (0.5 mL) wasadded KNO₃ (0.011 g, 0.11 mmol) and the resulting dark red solution wasstirred at room temperature for 65 h. The solution was then cooled in anice-bath and diluted with ice to a total volume of 5.0 mL. Theprecipitated solid was filtered, washed with water (2.0 mL) and driedunder vacuum to obtain 0.019 g (72%) crude product. It was purified asfollows. 0:016 g crude product was taken up in 1N NaOH (1.1 mL). Theinsoluble solid was centrifuged and the supernatant liquid was acidifiedwith conc. HCl to pH ˜2. The precipitated solid was filtered, washedwith water (1.0 mL), and dried under vacuum to furnish 0.010 g (38%)pure (purity by HPLC>97%) title compound as a cream colored powder, map.338-343° C. (decomposed); ¹H NMR (DMSO-d₆: δ 7.351 (s, 1H), 12.251 (soverlapped by a br s, 2H); Elemental analysis for C₈H₃BrClN3O₄ calcd: C,29.98%; H, 0.94%; N, 13.11%, found: C, 29.79; H, 0.77; N, 12.71.

Example 10

Preparation of 6-Bromo-7-Fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione

1-Bromo-2,5-difluoro-4-nitrobenzene:

To a stirred solution of 1-bromo-2,5-difluorobenzene (1.000 g, 5.181mmol, Aldrich, used as received) in conc. H₂SO₄ (8.0 mL) at 0° C, KNO₃(0.525 g, 5.19 mmol) was added in one lot. The resulting yellow solutionwas allowed to warm to 28° C. and stirred at 28° C. overnight. It wasthen poured into ice (80 g) and extracted with ethyl acetate (75 mL).The ethyl acetate was dried over anhydrous NaSO₄, removed under vacuum,and the resulting white solid was dried further under vacuum to afford1.102 g (89%) of the title compound as a white powder; m.p. 58-60° C.;¹H NMR (CDCl₃: δ 7.591 (dd, 1H, J_(1=9.6) Hz, J₂=5.4 Hz), 7.891 (t, 1H,J=6.9 Hz).

N-(5′-Bromo-4′-fluoro-2′-nitrophenyl)glycine sodium salt:

To a stirred solution of 1-bromo-2,5-difluoro-4-nitrobenzene (1.100 g,4.622 mmol, as prepared above) in DMF (11.0 mL) at 70° C., was added,dropwise, a solution of sodium glycinate (0.451 g, 4.65 mmol, Aldrich,used as received) in water (5.0 mL). The resulting solution was stirredovernight at 70° C. The solution was cooled to room temperature and thebright orange solid was filtered, washed with cold acetone (10 mL) anddried under vacuum to give 0.469 g (35%) of the title compound as abright orange powder; ¹H NMR (DMSO-d₆): δ 3.458 (d, 2H, J=3.9 Hz), 7.148(d, 1H, J=6.0 Hz), 7.944 (d, 1H, J=9.3 Hz), 8.740 (s, 1H).

6-Bromo-3,4-dihydro-7-fluoro-quinoxaline-2(1H)-one:

A solution of N-(5′-bromo-4′-fluoro-2′-nitrophenyl)glycine sodium salt(0.450 g, 1.54 mmol, as prepared above) and tin (II) chloride dihydrate(1.039 g, 4.605 mmol, Aldrich, used as received) in ethanol (7.0 mL) wasrefluxed for 30 min. It was then cooled to room temperature and solventwas removed under vacuum. The residue was diluted with water (15.0 mL)and basified with 10% Na₂CO₃ to pH ˜8. The resulting suspension wasextracted with ethyl acetate (100 mL). The ethyl acetate was dried overanhydrous NaSO₄ and removed under vacuum to yield 0.241 g (64%;)of thetitle compound as a yellow powder, m.p. 214-216° C.; ¹H NMR (DMSO-d₆): δ3.684 (s, 2H), 6.062 (s, 1H), 6.631 (d, 1H, J=9.3 Hz), 6.825 (d, 1H,J=6.6 Hz), 10.303 (s, 1H).

6-Bromo-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione:

To a stirred solution of6-bromo-3,4-dihydro-7-fluoro-quinoxaline-2(1H)-one (0.050 g, 0.20 mmol,prepared as above) in CF₃COOH (1.0 mL), excess fuming HNO₃ (0.10 mL) wasadded and the resulting red suspension was stirred overnight at 28° C.Solvent was removed under vacuum and the residue was diluted with water(2.0 mL). The precipitated solid was filtered, washed with water (2.0mL), and dried in a drying pistol (toluene reflux) to yield 0.034 g(55%) of pure (¹H NMR) title compound as a yellow powder; m.p. 323-327°C.; ¹H NMR (DMSO-d₆): δ 7.179 (d, 1H, J=9.0 Hz), 12.208 (s, 1H), 12.317(s, 1H); Elemental analysis for C₈H₃BrFN₃O₄ calcd. C, 31.60%; H, 0.99%;N, 13.82%, found C, 31.30%; H, 0.87%; N, 13.66%.

Example 11

Preparation of 7-Bromo-6-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione

1-Bromo-2,4-difluoro-5-nitrobenzene:

To a stirred solution of 1-bromo-2,4-difluorobenzene (0.512 g, 2.65mmol, Aldrich, used as received) in conc. H₂SO₄ (5.0 mL) at 0° C., KNO₃(0.275 g, 2.72 mmol) was added in one lot. The resulting solution wasallowed to warm to 28° C. and stirred at that temperature overnight. Itwas then poured into ice (50 g) and extracted with ethyl acetate (50mL). The ethyl acetate was dried over anhydrous Na₂SO₄, removed undervacuum and the resulting oil dried further under vacuum to afford 0.576g (91%) of the pure (¹H NMR) title compound as a light red oil; ¹H NMR(CDCl₃); δ 7.141 (dd, 1H, J₁=10.2 Hz, J₂=7.8 Hz), 8.375 (t, 1H, J=7.5Hz).

N-(4′-Bromo-5′-fluoro-2′-nitrophenyl)glycine andN-(2′-bromo-5′-fluoro-4′-nitrophenyl)glycine:

To a stirred solution of 1-bromo-2,4-difluoro-5-nitrobenzene (365 mg,1.53 mmol, as prepared above) in DMF (3.0 mL), was added dropwise, asolution of sodium glycinate (0.152 g, 1.57 mmol, Aldrich, used asreceived) in water (0.6 mL). The resulting suspension was stirred at 28°C. overnight. The solvent was removed under vacuum and the resultingslurry was cooled in an ice-bath. 1N HCl (1.5 mL) was added to it whichinstantly gave a yellow solid that was filtered and dried in a dryingpistol (toluene reflux) to give 0.175 g (39%) of a mixture of the titlecompounds in a ratio of 1.0:0.6 (¹H NMR) as a yellow powder; ¹H NMR(DMSO-d₆): δ 4.039 (d, 2H, J=6 Hz), 4.109 (d, 2H, J=5.4 Hz), 6.691 (d,1H, J=14.7 Hz), 6.900 (s, 1H), 7.005 (d, 1H, J=12.0 Hz), 8.219 (d, 1H,J=8.1 Hz), 8.337 (d, 1H, J=7.5 Hz), 8.494 (s, 1H). The separation of themixture was not feasible at this stage; hence, it was used as such forthe next reaction.

7-Bromo-3,4-dihydro-6-fluoro-quinoxaline-2(1H)-one:

A solution of a mixture of N-(4′-bromo-5′-fluoro-2′-nitrophenyl)glycineand N-(2′-bromo-5′-fluoro-4′-nitrophenyl)glycine (0.150 g, 0.512 mmol,as prepared above) and tin (II) chloride dihydrate (0.346 g, 1.53 mmol,Aldrich, used as received) in ethanol (3.0 mL) was refluxed for 30 min.It was then cooled to room temperature and the solvent was removed undervacuum. The residue was diluted: with water (10 mL) and basified withsaturated NaHCO₃ (3.0 mL) to pH ˜8. The resulting white suspension wasextracted with ethyl acetate (30 mL). The ethyl acetate was dried overanhydrous Na₂SO₄ and removed under vacuum to yield 0.050 g of crudeproduct, which was purified by precipitation from ethanol:water (1:1) togive 30 mg (24%) of the pure (¹H NMR) tide compound as a light yellowpowder, m.p. 172° C. (decomposed); ¹H NMR (DMSO-d₆): δ 3.73 (s, 2H),6.37 (s, 1H), 6.551 (d, 1H, J=10.2 Hz), 6.825 (d, 1H, J=6.6 Hz), 10.303(s, 1H).

7-Bromo-6-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione:

To a stirred solution of7-bromo-3,4-dihydro-6-fluoroquinoxaline-2(1H)-one (0.023 g, 0.094 mmol,prepared as above) in CF₃COOH (0.30 mL), excess fuming HNO₃ (0.015 mL)was added and the resulting red suspension was stirred overnight at 28°C. The red solution so obtained was cooled in an ice bath and dilutedwith water (2.0 mL). The precipitated solid was filtered, washed withwater (2.0 mL) and dried in a drying pistol (toluene reflux) to yield0.017 mg (60%) of the pure (¹H NMR) title compound as a brick redpowder; m.p. 316-321° C.; ¹H NMR (DMSO-d₆): δ 7.458 (d, 1H, J=6.3 Hz),12.006 (br s, 1H), 12.178 (s, 1H); Elemental analysis for C₈H₃BrFN₃O₄,calcd: C, 31.60%; H, 0.99%; N, 13.82%, found: C, 31.78%; H, 0.84%; N,13.49%.

Example 12

Preparation of 7-Bromo-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione

2-Bromo-5-fluoro-4-nitrotoluene:

To a stirred solution of 2-bromo-5-fluorotoluene (1.495 g, 7.909 mmol,Aldrich, used as received) in conc. H₂SO₄ (10.0 mL) at 0° C., KNO,(0.800 g, 7.91 mmol) was added in one lot. The resulting pale yellowsolution was warmed to 28° C. and stirred overnight at 28° C. It wasthen poured into ice (50 g) and extracted with ethyl acetate (75 mL).The ethyl acetate was dried over anhydrous Na₂SO₄, removed under vacuum,and the resulting oil dried further under vacuum to afford 1.832.g (98%)of the title compound as an oil; ¹H NMR (CDCl₃): δ 2.495 (s, 3H), 7.213(d, 1H, J₁=11.4 Hz), 8.268 (d, 1H, J₁=7.2 Hz).

N-(4′-Bromo-5′-methyl-2′-nitrophenyl)glycine sodium salt:

To a stirred solution of 2-bromo-5-fluoro-4-nitrotoluene (1.662 g, 7.102mmol, as prepared above) in DMF (7.0 mL) at 70° C., was added, dropwise,a solution of sodium glycinate (0.690 g, 7.11 mmol, Aldrich, used asreceived) in water (7.0 mL). The resulting suspension was stirredovernight at 70° C. The suspension was cooled to room temperature andthe red solid was filtered, washed with acetone, and dried under vacuumto give 0.932 g (45%) of the pure (¹H NMR) title compound as a redpowder; ¹H NMR (DMSO-d₆): δ 2.306 (s, 3H), 3.452 (d, 2H, J=3.96 Hz),6.874 (s, 1H), 8.125 (s, 1H), 8.779 (s, 1H).

7-Bromo-3,4-dihydro-6-methylquinoxaline-2(1H)-one: A solution ofN-(4′-bromo-5′-methyl-2′-nitrophenyl)glycine sodium salt (0.200 g, 0.692mmol, as prepared above) and tin (II) chloride dihydrate (0.468 g, 2.07mmol, Aldrich, used as received) in ethanol (2.0 mL) was refluxed for 30min. It was then cooled to room temperature. The precipitated solid wasfiltered and dried under vacuum to yield 0.064 g (38%) of the titlecompound as a yellow powder; ¹NMR (DMSO-d₆): δ 2.130 (s, 3H), 3.674 (s,21), 6.120 (s, 1H), 6.583 (s, 1H), 6.868 (s, 1H), 10.284 (s, 1H).

7-Bromo-6-methyl-5-nitroquinoxaline-2(1H),3(4H)-dione:

To a stirred suspension of7-bromo-3,4-dihydro-6-methyl-quinoxaline-2(1H)-one (0.028 g, 0.012 mmol,prepared as above) in CF₃COOH (0.30 mL), excess fuming HNO₃ (0.020 mL)was added and the resulting red solution was stirred overnight at 28° C.The suspension so obtained was cooled in an ice bath and diluted withwater (2.0 mL). The precipitated solid was filtered, washed with water(2.0 mL) and dried in a drying pistol (toluene reflux) to yield 0.014 g(40%) of the pure (¹H NMR) tide compound as a light yellow powder;m.p.>340° C.; ¹H NMR (DMSO-d₆): δ 2.227 (s, 311), 7.440 (s, 1H), 11.980(s, 1H), 12.143 (s, 1H); Elemental analysis for C₉H₆BrN₃O₄.0.45 H₂Ocalcd C, 35.07%; H, 1.96%; N, 13.63%, found C, 35.44%; H, 1.92%; N,13.23%.

Example 13

Preparation of7-Chloro-6-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione

1-Chloro-2,4-difluoro-5-nitrobenzene: To a stirred solution of1-chloro-2,4-difluorobenzene (0.829 g, 5.58 mmol, Aldrich, used asreceived) in conc. H₂SO₄ (8.0 mL) at 0° C., KNO₃ (0.565 g, 5.59 mmol)was added in one lot. The resulting solution was allowed to warm to 28°C. and stirred overnight at 28° C. It was then poured into ice (80 g)and extracted with ethyl acetate (75 mL). The ethyl acetate was driedover anhydrous Na₂SO₄, removed under vacuum, and the resulting oil wasdried further under vacuum to afford 1.007 g (93%) of the pure (¹H NMR)title compound as a light red oil; ¹H NMR (CDCl₃): δ 7.168 (dd, 1H,J₁=9.9 Hz, J₂=8.4 Hz), 8.238 (t, 1H, J=7.5 Hz).

N-(4′-Chloro-5′-fluoro-2′-nitrophenyl)glycine andN-(2′-chloro-5′-fluoro-4′-nitrophenyl)glycine sodium salt:

To a stirred solution of 1-chloro-2,4-difluoro-5-nitrobenzene (1.000 g,5.167 mmol, as prepared above) in DMF (10.0 mL), was added, dropwise, asolution of sodium glycinate (0.502 g, 5.17 mmol, Aldrich, used asreceived) in water (2.0 mL). The solution was stirred at 70° C. for 16 hand cooled to room temperature. The precipitated solid was filtered,washed with acetone (10 mL), and dried under vacuum to give 0.438 g(38%) of a red solid as a mixture of the title compounds in a ratio of1.0:0.3 as judged by ¹H NMR; ¹H NMR (DMSO-d₆): δ 3.474 (d, 2H, J=4.5Hz), 3.523 (d, 2H, J=3.9 Hz), 6.535 (d, 1H, J=14.7 Hz), 6.871 (d, 1H,J=12.3 Hz), 6.976 (s, 1H), 8.068 (d, 1H, J=7.8 Hz), 8.168 (d, 1H, J=7.8Hz), 8.867 (s, 1H). The separation of the mixture was not feasible atthis stage; hence, it was used as such for the next reaction.

7-Chloro-3,4-dihydro-6-fluoroquinoxaline-2(1H)-one:

A solution of a mixture of N-(4′-chloro-5′-fluoro-2′-nitrophenyl)glycinesodium salt and N-(2′-chloro-5′-fluoro-4′-nitrophenyl)glycine sodiumsalt (0.175 g, 0.704 mmol, as prepared above) and tin (II) chloridedihydrate (0.475 g, 2.11 mmol, Aldrich, used as received) in ethanol(3.5 mL) was refluxed for 30 min. It was then cooled to room temperatureand the solvent was removed under vacuum. The residue was diluted withwater (10 mL) and basified with saturated NaHCO₃ (3.0 mL) to pH ˜8. Theresulting white suspension was extracted with ethyl acetate (30 mL). Theethyl acetate was dried over anhydrous Na₂SO₄ and removed under vacuumto yield 0.041 g (29%) of the pure (¹H NMR) title compound as a lightyellow powder; m.p. 217-219° C. (decomposed); ¹H NMR (DMSO-d₆): δ 3.745(s, 2H), 6.37 (s, 1H), 6.587 (d, 1H, J=10.5 Hz), 6.741 (d, 1H, J=7.2Hz), 10.331 (s, 1H).

7-Chloro-6-fluoro-5-nitroquinoxaline-2(1H),3(4H)-dione:

To a stirred solution of7-chloro-3,4-dihydro-6-fluoro-quinoxaline-2(1H)-one (0.024 g, 0.12 mmol,prepared as above) in CF₃COOH (0.40 mL), excess fuming HNO₃ (0.02 mL)was added and the resulting red solution was stirred overnight at 28° C.Solvent was removed under vacuum and the residue was diluted with water(2.0 mL). The precipitated solid was filtered, washed with water (1.0mL), and dried in a drying pistol (toluene reflux) to yield 0.023 g(74%) of the pure (¹H NMR) title compound as a light yellow powder; m.p.308-310° C.; ¹H NMR (DMSO-d₆); δ 7.374 (d, 1H, J=6.9 Hz), 12.022 (br s,1H), 12.221 (s, 1H); elemental analysis for C₈H₃ClFN₃O₄ calcd C, 37.02%;H, 1.16%; N, 16.19%; found C, 37.03%; H, 1.19%; N, 15.33%.

Example 14

Preparation of 6-Azido-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione

To a solid mixture of 23 mg (0.094 mmol) of6,7-difluoro-5-nitroquinoxaline-2,3-dione and 7 mg (0.10 mmol) of sodiumazide was added 0.5 mL of DMSO-d₆ and the mixture was shaken in a vortexfor 10 s. Some white insoluble material was observed. One drop of D₂Owas added and shaken for 10 s. Again, some white solid was observed. ¹HNMR, 7.109 (1, d, J=12.0). The solution was added to 3 mL of ice-water.The yellow precipitate was filtered, washed with water, and dried toleave a yellow solid (21 mg, 84%); mp 255-257° C.; ¹H NMR (DMSO-d₆),7.166 (1, d, J=11.8), 12.2-12.1 (m, 2). ¹⁹F NMR (C₆F₆ as reference,−162.9 ppm), −130.5 (mb). IR (KBr), 2154, 1753, 1540, 1359, 1308 cm⁻¹.MS, 266 (M⁺, 1), 238 (M⁺−N₂, 100), 178 (90), 150 (40).

Example 15

Preparation of 6-Amino-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione

To a solution of 20 mg (0.082 mmol) of6,7-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione in 0.5 m; ofDMSO-d₆ was added 2 drops of 30% ammonium hydroxide and the solution washeated at 80° C. for 24 h. To the mixture was added one more drop of 30%ammonium hydroxide and it was heated at 80° C. for 10 h. The mixture wasadded to 4 mL of water and then acidified with 2 N HCl to pH=1. Theprecipitate was filtered, washed with water, and dried to leave a yellowsolid (15 mg, 76%); mp 250° C. (dec.); ¹H NMR (DMSO-d₆), 7.202 (1, d,J=11.5), 7.232 (sb, 2), 11.15 (mb, 1), 12.0 (mb, 1). ¹⁹F NMR, −133.84(d, J=11.6). MS, 240 (M⁺, 100), 212 (10), 177 (10), 166 (20). HRMS,Calcd for CH₅FN₄O₄ 240.0290, found 240.0294.

Example 16

Preparation of7-Fluoro-6-methoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione

To a solution of 21 mg (0.086 mmol) of6,7difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione in 0.5 mL ofDMSO-d₆ was added 16 mg (0.29 mmol) of sodium methoxide. The solutionwas shaken in a vortex for 10 s and kept at room temperature for 3 h. Itwas then added to 3 mL of water and acidified with 2 N HCl to pH=4. Theprecipitate was filtered, washed with water, and dried to leave a yellowsolid (17 mg, 77%); mp 290-292° C.; ¹H NMR (DMSO-d₆), 3.899 (s, 3),7.163 (1, d, J=11.6), 11.97 (mb, 1), 12.137 (s, 1). ¹⁹F NMR, −134.48(mb). MS, 255 (M⁺, 100), 243 (15), 179 (16), 151 (40). HRMS, Calcd forC₉HFN₃O₅ 255.0287, found 255.0308.

Example 17

Preparation of 7-Fluoro-6-ethoxy-5-nitro-1,4dihydroquinoxaline-2,3-dione

A solution of 24 mg (0.098 mmol) of6,7difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione and 24 mg (0.33mmol) of sodium ethoxide (96%) in 0.5 mL of DMSO-d₆ was kept at roomtemperature for 5 h. The red solution was added to 3 mL of water andacidified with 2 N HCl to pH =4. The precipitate was filtered, washedwith water, and dried to leave a yellow solid (24 mg, 91%); mp 293-295°C.; ¹H NMR (DMSO-d₆), 1.239 (t, 3, J=7.0), 4.141 (q, 2, J=7.2), 7.152(1, d, J=11.7), 11.975 (s, 1), 12.135 (s, 1). ¹⁹F NMR, −134.06 (mb). MS,269 (M⁺, 90), 241 (100), 195 (70). HRMS, Calcd for C₁₀H₈FN₃O₅269.0443,found 269.0454.

Example 18

Preparation of7-Fluoro-6-hydroxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione

A mixture of 24 mg (0.098 mmol) of6,7-difluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione and 19 mg (0.47mmol) of sodium hydroxide in 0.6 mL of D₂O was heated at 105° C. for 24h. The mixture was added to 2 mL of water and acidified with 2 N HCl topH=1. The precipitate was filtered, washed with water, and dried toleave a red solid (19 mg, 80%); mp>360° C.; ¹H NMR (DMSO-d₆), 7.101 (1,d, J=10.9), 11.10 (mb, 1), 11.70 (mb, 1), 11.99 (s, 1). ¹⁹F NMR, −137.4(mb). MS 242 (M++1, 100), 195 (50), 152 (20), 140 (25). HRMS, Calcd forC₈H₄FN₃O₅ 241.0131, found 241.0110.

Example 19

Preparation of 5-Azido-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione

A mixture of 45 mg (0.18 mmol) of5-amino-6,7-dichloro—1,4-dihydroquinoxaline-2,3-dione in 1 mL ofconcentrated H₂SO₄ (97%) was stirred in an ice-bath for 1 h. To theresulting yellow solution was added, dropwise, a solution of 70 mg (1.0mmol) of NaNO₂ in 0.6 ML of H₂O and the solution was stirred in anice-bath for 3 h. To the resulting red solution was added a solution of98 mg of NaN₃ in 0.6 mL of H₂O and it was stirred for 1 h. To themixture was added a solution of 101 mg of NaN₃ in 0.6 mL of H₂O and themixture was stirred overnight. The mixture was filtered, washed withwater, and dried to leave a yellow solid 44 mg (88%); mp 130° C.(decomposed); ¹H NMR (DMSO-d₆), 7.148 (s,1), 11.75 (mb,1), 12.09 (s,1).IR (KBr), 2120, 1700 cm⁻¹.

Example 20

Preparation of 6-Azido-5,7-dichloro-1,4-1,4-dihydroquinoxaline-2,3-dione

A mixture of 10 mg (0.040 mmol) of 6-amino-5,7-dichloro-1,4dihydroquinoxaline-2,3-dione in 0.5 mL of concentrated H₂SO₄ (97%) wasstirred in an ice-bath for 1 h. To the resulting yellow solution wasadded, dropwise, a solution of 30 mg (0.43 mmol) of NaNO₂ in 0.3 mL ofH₂O and the solution was stirred in an ice bath for 2 h. To theresulting red solution was added a solution of 40 mg of NaN₃ in 0.3 mLof H₂O and it was stirred for 2 h. The mixture was diluted with 2 mL ofH₂O and stirred overnight. The mixture was filtered, washed with water,and dried to leave an almost colorless solid 10 mg (90%); mp 150° C.(decomposed); ¹H NMR (DMSO-d₆), 7.153 (s,1), 11.602 (s,1), 12.02 (s,1).IR (KBr), 2127, 1720 cm⁻¹.

Example 21

Preparation of 6,7-Dimethyl-1,4-dihydroquinoxaline-2,3-dione

A mixture of 2.72 g (2.0 mmol) of 4,5-dimethyl-1,2-phenylenediamine and1.92 g (21.3 mmol) of oxalic acid in 30 mL of 2N HCl was refluxed for2.5 h and cooled to room temperature. The mixture was diluted with 20 mLof H₂O, filtered, washed with water, and dried to leave a pale-brownsolid 3.57 g (94%); mp>250° C.; ¹H NMR (DMSO-d₆,2.161 (s,6), 6.869(s,2), 11.78 (s,2).

Example 22

Preparation of 6,7-Dimethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione

To a solution of 1.90 g (10.0 mmol) of6,7-dimethyl-1,4-dihydroquinoxaline-2,3-dione in 25 mL of H₂SO₄ (97%)kept in an ice bath was added dropwise 1.2 mL of HNO₃ (69-70%) and thesolution was stirred ;at room temperature for 48 h. It was poured into300 mL of ice-water and stirred for 10 min. The mixture was filtered,washed with water, and dried to leave a yellow solid 1.40 g (60%). Thesolid was purified by DMSO/H₂O precipitation followed by NaOH/HClprecipitation to leave 0.85 g of a yellow solid; mp>250° C.; ¹H NMR(DMSO-d₆), 2.079 (s,3), 2.246 (s,3), 7.048 (s,1), 11.770 (s,1), 12.030(s,1). HRMS calcd. for C₁₀H₉N₃O₄ 235.0588; found 235.0584.

Example 23 Preparation of7-Bromo-5-ethyl-1,4-dihydroquinoxaline-2,3-dione

A mixture of 1,2-diamino-4-bromo-6-ethylbenzene (40 mg, 0.14 mmol) andoxalic acid dihydrate (25 mg, 0.20 mmol, used as received) in 4 N HCl(1.5 mL) was refluxed at 120-5° C. for 3 h, then cooled to roomtemperature. The mixture was centrifuged and the liquid layer wasremoved. The yellow solid was washed with cold water (2×1 mL), collectedby filtration, and dried at 60° C. under reduced pressure for 2 h,affording 40 mg of crude product (80%) as a yellow powder. The crudeproduct was dissolved in 1N NaOH (2 mL) and filtered. The filtrate wasacidified to pH=5, affording 12 mg of pure title compound; mp: >350° C.(dec. from 300° C.), IR (KBr, cm⁻¹):3410, 3164, 2919, 1740, 1705, 1600.¹H NMR (DMSO-d₆): 1.099 (t, 3 H; J=6 Hz), 2.741 (q, 2 H, J=7.2 Hz),7.117, (d, 1 H, J=1.8 Hz), 7.138 (d, 1 H, J=1.8 Hz); 11.343 (s, 1H);11.958 (s, 1H). HRMS: calcd for C₁₀H₉BrN₂O₂ (M⁺) m/z: 267.9846; found267.9853.

Example 24

Preparation of 5,7-Dimethyl-1,4-dihydro-2,3-quinoxalinedione

1,2-Diamino-4,6-dimethylbenzene.

A mixture of 4,6-dimethyl-2-nitroaniline (1.66 g, 10.0 mmole) and 10%Pd/C (200 mg) in ethanol (35 mL) was hydrogenated for 2 h at roomtemperature under 25 psi H₂. The catalyst was removed by filtration withcelite and the solvent was removed by rota-evaporation to give 1.300 g(96%) of 1,2-amino-4,5-dimethylbenzene as a brown solid. ¹H NMR (CDCl₃):6.449 (s, 1H), 6.469 (s, 1H), 3.327 (br, 2H), 3.259 (br, 2H), 2.190 (s,3H), 2.159 (s, 3H).

5,7-dimethyl-1,4-dihydro-2,3quinoxalinedione.

A mixture of 1,2-diamino-4,6-dimethylbenzene (424 mg, 3.11 mmole) andoxalic acid dihydrate (432 mg, 3.43 mmole, used as received) in 4N HCl(20 mL) was refluxed at 120-5° C. for 3 h, then cooled to roomtemperature. The mixture was centrifuged and the supernatant wasremoved. The yellow solid was washed with cold water (2×2 mL), collectedby filtration, and dried in vacuo for 2 h, affording 516 mg of crude5,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione (87%) as a yellow powder.The crude product was taken up in 1 N NaOH (10 mL) and filtered. Thefiltrate was acidified to pH =3, affording the pure title compound (490mg) as a light yellow powder; mp:345-347° C. (dec); IR (KBr, cm⁻¹):3460,3190, 2986, 1716, 1709, 1630. ¹H NMR (DMSO-d₆): δ11.858 (s, 1H), 11.173(s, 1H), 6.766 (s, 1H), 6.745 (s, 1H), 2.280 (s, 3), 2.211 (s, 3). HRMS:calcd for C₁₀H₁₀(N₂O₂ (M⁺) m/z: 190.0714; found: 190.0744.

Example 25

Preparation of 1,4-Dihydrvbenzo[g]quinoxaline-2,3-dione

A mixture of 603 mg (3.81 mmol) of 2,3-naphthalenediamine and 382 mg(4.24 mmol) of oxalic acid in 5 mL of 2N HCl was refluxed for 3 h andcooled to room temperature. The mixture was filtered, washed with water,and dried to leave a brown solid 803 mg (99%); mp >250° C.; 1H NMR(DMSO-d₆), 7.382 (dd, 2, J=3.16, 6.17), 7.525 (s, 2), 7.815 (dd, 2,J=3.22, 6.18), 12.088 (s, 2).

Example 26

Preparation of 5-Methyl-1,4-dihydro-2,3-quinoxalinedione

To a stirred solution of 2,3-diaminotoluene (0.498 g, 0.407 mmol,Aldrich) in 2 N HCl (6 mL, 12 mmol), oxalic acid dihydrate (0.520 g,0.412 mmol, Fisher) was added in one portion. The resulting deep purplesolution was refluxed for 13 h, to give a purple suspension. This wascooled to 25° C., filtered, washed with water (5 mL), and dried in vacuo(0.1 mm Hg) to give a purple powder. This was taken up in 2 N NaOH (25mL, 50 mmol), giving a brown solution, which was filtered. The brownfiltrate was acidified to pH 1.0 by addition of 2 N HCl (25 mL, 50mmol), resulting in a tan suspension. Filtration of the suspension andwashing of the filter cake with water (5 mL) gave the title compound asa tan powder (551 mg, 64%); mp 325° C. (the block was preheated to 320°C.); ¹H NMR (DMSO-d₆) δ 2.26 (s, 3H), 6.86-7.01 (m, 3H), 11.6 (broad,2H).

Example 27

Preparation of 6-Methyl-1,4-dihydro-2,3-quinoxalinedione

To a stirred solution of 3,4-diaminotoluene (0.302 g, 0.247 mmol,Aldrich) in 2 N HCl (4 mL, 8 mmol), oxalic acid dihydrate (0.330 g,0.261 mmol, Fisher) was added in one portion. The resulting deep purplesolution was refluxed for 13 h, to give a purple suspension. This wascooled to 25° C., filtered, washed with water (5 mL), and dried in vacuo(0.1 mm Hg) to give a blue-grey powder. This was taken up in 2 N NaOH(34 mL, 68 mmol), giving a brown solution, which was filtered. The brownfiltrate was acidified to pH 1.0 by addition of 2 N HCl (42 mL, 84mmol), resulting in a tan suspension. Filtration of the suspension andwashing of the filter cake with water (5 mL) gave the title compound asa grey powder (225.5 mg, 43%); mp 318° C. (the block was preheated to300° C.); ¹H NMR (DMSO-d₆) δ 2.32 (s, 3H), 6.90-6.99 (m, 3H), 11.22 (s,1H), 11.90 (s, 1H).

Example 28

Preparation of 7-Methyl-nitro-1,4-dihydro-2,3-quinoxalinedione

Methyl-3-nitro-1,2-phenylenediamine.

The procedure of Gillespie et al., J. Org. Chem. 25:942 (1960) wasadopted as follows. A dark black solution of 4-methyl-2,6-dinitroaniline(0.100 g, 0.561 mmol, Aldrich, used as received) in 6.66% aq. (NH₄)₂S(3.3 mL, prepared from 20% aq.(NH₄)₂S solution supplied by Aldrich) andethanol (3.5 mL) was refluxed for 45 min. It was then cooled to 28° C.and the solvents were removed as much as possible under vacuum (insidethe hood to avoid the stench of ammonium sulfide). The slurry soobtained was diluted with water (10 mL) and the -resulting red solid wasfiltered and dried under vacuum to obtain 0.072 g (85%) solid as a redpowder, which was used as such for the next reaction. ¹H NMR(acetone-dip: 2.205 (s, 3H), 6.342 for s, 2H), 6.622 (s, 1H), 7.571 (s,1H).

7-Methyl-5-nitro-1,4-dihydro-2,3-quinoxalinedione.

A suspension of 5-methyl-3-nitro-1,2-phenylenediamine (0.050 g, 0.30mmol) and oxalic acid (0.038 g, 0.30 mmol) in 2N HCl (1.6 mL) wasrefluxed for 2.5 h during which time it first formed a solution and thena suspension. The suspension was then cooled to 28° C. and theprecipitated solid was filtered, washed with water (5 mL), and driedunder vacuum to afford 30 mg (45%) of product as a light yellow powder.It was purified by the base-acid treatment as follows. All of the crudeproduct was taken up in 1N NaOH (3.3 mL) and swirled at 90° C. todissolve most of the solid. It was then filtered hot and the filtratewas cooled in an ice-bath and acidified with concentrated HCl to pH ˜2.The precipitated solid was filtered and dried under vacuum to obtain0.025 g (38%) of the pure title compound as a light yellow powder; m.p.319°-323° C. (decomposed); ¹H NMR (DMSO-d₆): 2.352 (s, 31, 7.271 (s,1H), 7.75 (s, 1H), 11.044 (s, 1H), 12.299 (s, 1H); elemental analysisfor C₉H₇N₃O₄.0.35 H₂O calcd: C, 47.52%; H, 3.10%; N, 18.47%; found: C,47.82%; H, 3.03%; N, 18.06%.

Example 29

Preparation of7-Fluoro-6-methyl-5-nitro-1,4-dihydro-2,3-quinoxalinedione

2,5-Difluoro-4-nitrotoluene.

To a stirred solution of 2,5-difluorotoluene (0.544 g, 4.25 mmol,Aldrich, used as received) in conc. H₂SO₄ (5.0 mL) at 0° C., KNO₃ (0.430g, 4.25 mmol) was added in one portion. The resulting pale yellowsolution was warmed to 28° C. and stirred at that temperature overnight.It was then poured into ice (25 g) and extracted with ethyl acetate (40mL). The extract was dried over Na₂SO₄ and evaporated to afford 0.555 g(91%) of the title compound as a light red oil; ¹H NMR (CDCl₃): 2.369(d, 3H, J=1.8 Hz), 7.127 (dd, 1H, J₁=8.1 Hz, J₂=6.0 Hz), 7.734 (dd, 1H,J₁=8.4 Hz, J₂=6.3 Hz).

N-(4-Fluoro-5-methyl-2,nitrophenyl)glycine sodium salt.

To a stirred solution of 2,5-difluoro-4-nitrotoluene (0.550 g. 3.18mmol) in DMF (5.0 mL) was added dropwise a solution of sodium glycinate(0.308 g, 3.18 mmol, Aldrich, used as received) in water (1.0 mL). Theresulting suspension was stirred at 28° C. overnight. The solid wasfiltered and dried under vacuum to give 0.168 g (23%) of crude product.It was purified as follows. 0.150 g material was boiled in ethyl acetate(5.0 mL) and filtered while hot to give 0.134 g (18%) of the pure (¹HNMR) title compound as a yellow powder. ¹H NMR (DMSO-d₆): 2.213 (s, 3H),3.428 (d, 2H, J=3.9 Hz), 6.765 (d, 1H, J=6.9 Hz), 7.700 (d, 1H, J=10.5Hz), 8.728 (s, 1H)

7-Fluoro-3,4-dihydromethylquinoxaline-2(1H)-one. A mixture ofN-(4-fluoro-5-methyl-2-nitrophenyl)glycine sodium salt(0. 125 g, 0.548mmol) and tin (II) chloride dihydrate (0.370 g, 1.64 mmol, Aldrich, usedas received) in ethanol (3.0 mL) was refluxed for 30 min. It was thencooled to room temperature and the solvent was removed under vacuum. Theresidue was diluted with water (4.0 mL) and basified with saturatedNaHCO₃ to pH ˜8. The resulting. suspension was extracted with ethylacetate (30 mL). The extract was dried over Na₂SO₄ and evaporated toyield 0.032 g of the tide compound as a yellow powder, which was usedfor the next reaction; ¹H NMR (DMSO-d₆): 2.024 (s, 3H), 3.607 (s, 2H),5.751 (s, 1H), 6.453 (d, 1H, J=9.9 Hz), 6.456 (d, 1H, J=8.1 Hz), 10.162(s, 1H).

7-Fluoro-6-methyl-5-nitro-1,4-dihydro-2,3-quinoxalinedione.

To a stirred solution of7-fluoro-3,4-dihydro-6-methylquinoxaline-2(1H)-one (0.023 g, 0.094 mmol)in CF₃COOH (0.30 mL), excess fuming HNO₃ (0.020 mL) was added and theresulting red suspension was stirred overnight at 28° C. The redsolution so obtained was cooled in an ice bath and diluted with water(2.0 mL). The precipitate was filtered, washed with water (2.0 mL), anddried in a drying pistol (toluene reflux) to yield 0.020 g (66%) of thepure title compound as a yellow powder; m.p. 308-311° C.; ¹H NMR(DMSO-d₆): 2.106 (s, 3H), 7.058 (d, 1H, J=9.9 Hz), 11.769 (bs, 1H),12.172 (s, 1H).

Example 30

Preparation of 6-Chloro-5-cyano-7-nitro-1,4-dihydroquinoxaline-2,3-dione

To a stirred solution of 2,6-dichloro-3-nitrobenzonitrile (3.935 g,18.13 mmol, Lancaster, used as received) in DMF (25 mL) at 70° C., anaqueous solution of sodium glycinate (1.760 g, 18.13 mmol, Aldrich, usedas received) in water (25.0 mL) was added dropwise. The resultingsolution was stirred at 70° C. for 48 h. The suspension was cooled toroom temperature and the precipitated yellow solid was filtered, washedwith chloroform (20 mL), and dried under vacuum to furnish 2.020 g (44%)of pure (¹H NMR) N-(3′-chloro-2′-cyano-6′-nitro)phenylglycine as ayellow powder. ¹H NMR (DMSO-d₆): δ 3.888 (d, 2H, J=3.9 Hz), 6.857 (d,1H, J=9.0 Hz), 8.283 (d, 1H, J=9.3 Hz), 9.572 (s, 1H).

A suspension of N-(3′-chloro-2′-cyano-6′-nitro)phenylglycine (2.000 g,7.824 mmol, as prepared above) and tin (11) chloride dihydrate (6.000 g,26.59 mmol, Aldrich, used as received) in ethanol (40 mL) was refluxedfor 30 min. The resulting suspension was cooled to room temperature andthe solid was filtered, washed with ethanol (20 mL), and dried undervacuum to give 1.261 g (78%) of pure (¹H NMR)6chloro-5-cyano-3,4-dihydroquinoxaline-2(1H)-one as a light yellowsolid. ¹H NMR (DMSO-d₆): δ 3.668 (s, 2H), 6.719 (d, 1H, J=8.4 Hz), 6.843(d, 1H, J=8.1 Hz), 6.912 (s, 1H), 10.682 (s, 1H).

To a suspension of 6-chloro-5-cyano-3,4-dihydroquinoxaline-2(1H)-one(0.096 g, 0.46 mmol, as prepared above) in CF₃COOH (1.0 mL), fumingnitric acid (0.40 mL) was added so that it formed a dark red solution.(A lesser amount of fuming nitric acid created a suspension that gave amixture of partially oxidized and fully oxidized products). Theresulting solution was then stirred overnight at room temperature. Thevolatiles were removed under vacuum and the residue was diluted withwater (3.0 mL). The yellow solid was filtered, washed with water (2.0mL), and dried under vacuum to furnish 0.090 g (73%) of the pure CH NMR)title compound as a yellow powder; m.p. 327-331° C.; ¹H NMR (DMSO-d₆): δ7.950 (s, 1H), 12.381 (s, 1H); IR (KBr, cm⁻¹): 3501, 3452, 3157, 2259,1740, 1712, 1635, 1607, 1550, 1396, 1340; elemental analysis forC₉H₃ClN₄O₄ calcd: C, 40.55%; H, 1.13%; N, 21.02%; found C, 40.57%; H,1.11%; N, 20.84%.

Example 31

Preparation of 5-Cyano-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione

A suspension of 6-chloro-5-cyano-7-nitroquinoxaline-2(1H), (3(4H)-dione(0.100 g, 0.38 mmol, as prepared above) and tin (II) chloride dihydrate(0.508 g, 2.25 mmol, Aldrich) in ethanol (4.0 mL) was refluxed for 24 h.The resulting suspension was then cooled to room temperature and theyellow solid was filtered, washed with ethanol (2.0 mL), and dried undervacuum to obtain 0.078 g (88%) of7-amino-6-chloro-5-cyanoquinoxaline-2(1H),3(4H)dione as a yellow powder;¹H NMR (DMSO-d₆): δ 5.762 (br s, 2H), 6.818 (s, 1H), 11.69 (s, 1H),12.03 (s, 1H).

To a stirred solution of7-amino-6-chloro-5-cyanoquinoxaline-2(1H),3(4H)dione (0.035 g, 0.15mmol, as prepared above) in concentrated HCl (1.5 mL) at 0° C., anaqueous solution of NaNO₂ (0.060 g, 0.87 mmol) in water (0.20 mL) wasadded and the resulting turbid solution was stirred in an ice bath for 2h. A solution of CuCl (0.100 g, 1.01 mmol) in concentrated HCl (1.0 mL)was added to it while cooling the flask in an ice bath. Instantevolution of N₂ ensued and the resulting dark green suspension wasstirred at 0° C. for 2 h. Water (1.0 mL) was added to it followed bystirring overnight at room temperature. To the light green suspension soformed, water (4.0 mL) was added with further stirring at roomtemperature for 1 h. The precipitated solid was filtered, washed withwater (2.0 mL), and dried under vacuum to furnish 0.029 g (77%) of thetitle compound as a cream colored solid; m. p. 327-335° C. (decomposed);¹H NMR (DMSO-d₆): δ 7.463 (d, 1H, J=1.2 Hz), 12.306 (s, 2H). Thecoupling of aromatic signal disappeared upon addition of 3 drops ofmethanol-d₄ indicating that the aromatic proton had coupled with periN—H proton; IR (KBr, cm⁻¹); 3574, 3479, 2237, 1737, 1710, 1392, 1271,1183.

Example 32

Preparation of7-Chloro-6-methoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione

To a stirred solution of7-Chloro-6-fluoro-5-nitroquinoxaline-2(1H),3(4H)dione (0.100 g, 0.385mmol, as prepared above) in DMSO (1.0 mL) at room temperature, sodiummethoxide (0.125 g, 2.31 mmol, Mallinckrodt) was added in one port. Theresulting dark red solution was stirred overnight at room temperate. Itwas then diluted with water (5.0 mL) and acidified with concentrated HCl(6 drops) to pH ˜2. The precipitated solid was filtered, washed withwater (5.0 mL), and dried under vacuum to obtain 0.116 g (110%) of pure(¹H NMR) title compound as a yellow powder; m. p. darkens at 316° C.; ¹HNMR (DMSO-d₆): δ 3.821 (s, 3H), 7.275 (s, 1H), 12.079 (s, 1H) 12.121 (s,1H). The NMR also indicates the presence of DMSO (reaction solvent).

Example 33

Preparation of 6,7-Dimethoxy-1,4-dihydroquinoxaline-2,3-dione (10)

4,5-Dimethoxy-1,2-dinitrobenzene (8). The procedure of Wulfman andCooper, Synthesis 924 (1978), was adopted for this reaction.1,2-Dimethoxybenzene 7 (20.8 g, 0.15 mol; Aldrich) was added dropwiseover 30 min. to vigorously stirred 70% nitric acid (175 mL) undernitrogen. The temperature was kept below 50° C. during the addition.Shortly after the addition was complete, a yellow solid precipitated.The mixture was then heated to 70-80° C. for about 2 h (until evolutionof NO₂ ceased). The mixture was allowed to cool below 40° C., thenpoured into ice/water (1000 mL) and filtered, by suction filtration. Theyellow solid was slurried in saturated sodium bicarbonate (500 mL)overnight. The crude product was isolated by suction filtration and thenpurified by recrystallization from ethyl alcohol (1000 mL). The titlecompound was obtained as fine yellow needles (23 g, 67%). mp 128-130° C.(lit. mp 127-128° C.; Frisch and Bogert, J. Org. Chem. 8:331 (1943)) ¹HNMR (CDCl₃, 300 MHz) δ 4.016 (s, 6H), 7.337 (s, 2H).

6,7-Dimethoxy-1,4-dihydroquinoxaline-2,3-dione (10).

4,5-Dimethoxy-1,2-dinitrobenzene 8 (912 mg, 4.00 mmol) was dissolved inethyl acetate (30 mL). To this solution was added 10% Pd/C (228 mg, 20%;Aldrich). The mixture was then stirred at room temperature under apressure of 40 psi (H₂) for 14 hr. The catalyst was removed through acolumn of Celite (5 g) and washed with ethyl acetate (3×15 mL) undernitrogen. The filtrates were combined and the solvent was removed togive the diamine 9 as a nearly colorless solid. The ¹H NMR spectrum wasconsistent with the assigned structure. Diamine 9 was dissolved in 4 Nhydrochloric acid (7 mL), and oxalic acid dihydrate (504 mg, 4.00 mmol;Fisher) was added to this solution in one portion with stirring underN₂. The mixture was refluxed at 130-5° C. (oil bath) for 3 hr. A yellowsolid precipitated, which was collected by suction filtration and driedin vacuo overnight, giving 725 mg (82% based on compound 8) of the titlecompound as a pale yellow solid; mp 345-346° C.; ¹H NMR (CDCl₃, 300 MHz)δ 3.677 (s, 3H), 6.681 (s, 2H), 11.695 (s, 2H). EIMS m/e 222 (100, M⁺).(HPLC purity 99%)

Example 34

Preparation of 6,7-Methylenedioxy-1,4-dihydroquinoxaline-2,3-dione (15)

4,5-Methylenedioxynitrobenzene (12).

1,3-Benzodioxole (13.33 g, 109 mmol; Aldrich) was added dropwise tostirred, cold (−5-0° C.) concentrated nitric acid (150 mL) at a ratesuch that the temperature did not rise above 0° C. over 90 min. in anice/salt bath. After the addition was complete, the mixture was stirredat 0° C. for another 3 hr and was then poured into ice/water (750 mL).The product was isolated by filtration, washed with cold water until thewashings were no longer acidic (6×50 mL), and then with saturatedaqueous sodium bicarbonate (50 mL), giving 17.23 g (95%) of the titlecompound as a yellow solid; mp 147-148° C. (lit. mp 147° C.; Perkin etal., J. Chem. Soc. 94:1979 (1909)); ¹H NMR (CDCl₃, 300 MHz) δ 6.142 (s,2H), 6.868 (d, 1H, J=8.4 Hz), 7.667 (d, 1H, J=1.5 Hz), 7.894 (d, 1H,J=6.9 Hz).

4,5-Methylenedioxy-1,2-dinitrobenzene (13).

4,5-Methylenedioxynitrobenzene 12 (17.23 g, 103 mmol) was ground to afine powder and added in one portion to a stirred mixture of 70% (conc.)nitric acid (125 mL; Baker) and 90% (fuming) nitric acid (125 mL; Baker)at −15° C. Stirring was continued at −5° C. to 0° C. for another 3 hr.The mixture was then poured into ice/water (1250 mL). The product wasisolated by filtration, washed with cold water until the washings are nolonger acidic (6×50 mL), and then with saturated aqueous sodiumbicarbonate (50 mL), giving 19.1 g (87%) of the title compound as ayellow solid. Mp 99-100° C. (lit. mp 101° C.; Hughes and Ritchie, Aust.J. Chem. 7:104 (1954)) ¹H NMR (CDCl₃, 300 MHz) δ 6.270 (s, 2H), 7.303(s, 2H).

6,7-Methylenedioxy-1,4-dihydroquinoxaline-2,3-dione (15).(4,5-Methylenedioxy-1,2-dinitrobenzene 13 (1.37 g, 6.47 mmol) wasdissolved in ethyl acetate (20 mL). To this solution was added 10% Pd/C(343 mg, 20%; Aldrich). The mixture was then stirred at room temperatureunder a pressure of 40 psi (H₂) for 14 hr. The catalyst was removedthrough a column of Celite (5 g) and washed with ethyl acetate (3×15 mL)under nitrogen. The filtrates were combined and the solvent was removedto give the diamine 14 as a nearly colorless solid. The ¹H NMR spectrumwas consistent with the assigned structure. Diamine 14 was dissolved in4 N hydrochloric acid (7 mL), and oxalic acid dihydrate (983 mg, 6.47mmoL) was added to this solution in one portion with stirring under N₂.The mixture was refluxed at 130-5° C. (oil bath) for 3 hr. Ayellow-solid precipitated, which was collected by suction filtration anddried in vacuo overnight, giving 1.18 g (88.7% based on compound 13) ofthe title compound as a brown solid; mp >360° C.; ¹H NMR (DMSO-d₆, 300MHz) δ 5.998 (s, 2H), 6.775 (s, 2H), 11.695 (s, 2H). EIMS m/e 206 (100,M⁺). (HPLC purity 98%).

Example 35

Preparation of 6,7-diethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione (26)

1,2-Diethyl-4-nitrobenzene (16), 1,2-Diethyl-3-nitrobenzene (17), and4,5-Diethyl-1,3-dinitrobenzene (18).

The procedure of Lambooy, J.P., at J. Am. Chem. Soc. 71:3756 (1949) wasadopted for this reaction. A mixture of fuming nitric acid (8.75 mL;Baker) and glacial acetic acid (4.5 mL) was cooled to 10° C. To thismixture, 1,2-diethylbenzene 15 (2.5 g, 18.6 mmol;

Aldrich) was added dropwise at such a rate as to maintain thetemperature at 10-20° C. with vigorous stirring over 30 min. After theaddition was complete, the reaction was allowed to stir at 15° C. foranother 1 h. The reaction mixture was then poured into ice-water (50mL). The nitro compounds were extracted with ether (4×20 mL). Thecombined ether extract was washed with water (3×7.5 mL), 10% sodiumhydroxide aqueous solution (2×8 mL), and water (2×7.5 mL). The etherextract was dried over sodium sulfate. The solvent was evaporated togive a residue, which was purified by preparative TLC (Hexanes:EtOAc=8:2), giving the mono-nitro compounds 16 and 17 (1.90 g, 57%) as ayellow oil (R_(f)=0.8), accompanied by the dinitro compound 18 (0.30 g,7%) as a brown oil (R_(f)=0.5). ¹H NMR (CDCl₃, 300 MHz): δ (16): 1.220(t, 6H, J=7 Hz), 2.725 (q, 4H, J=7 Hz), 7.728 (d, 1H, J=6 Hz), 7.986 (d,1H, J=6.Hz), 8.034 (s, 1H). (17): 1.220 (t, 6H, J=7 Hz), 2.725 (q, 4H,J=7 Hz), 7.242 (m, 1H), 7.378 (d, 1H, J=7.5 Hz), 7.550 (d, 1H, J=7.5Hz). (18): 1.231 (t, 6H, J=7 Hz), 2.655 (q, 4H, J=7 Hz), 8.044 (s, 1H),10.636 (s, 1H).

3,4-Diethylaniline (19) and 2,3-Diethylaniline (20).

A mixture of 1,2-diethyl-4-nitrobenzene (16) and1,2-diethyl-3-nitrobenzene (17) (1.4 g, 7.82 mmol) was dissolved inethyl acetate (20 mL). To this solution was added 10% Pd-C (350 mg, 20%;Aldrich). The mixture was agitated at room temperature under a pressureof 35 psi (H) for 10 h. The catalyst was removed through a column ofCelite (5 g) and washed with ethyl acetate (3×15 mL). The filtrates werecombined and evaporated in vacuo to give a residue, which was separatedby preparative TLC (Hexanes: EtoAc=8:2), giving 3,4-diethylaniline (19)(0.822 g, 71%) as a pale yellow oil (R_(f)=0.7) and 2,3-diethylaniline(20) (0.219 g, 19%) as a brown oil (R_(f)=0.6). ¹H NMR (CDCl₃,300 MHz):δ (19): 1.184 (t, 6H, J=7 Hz), 2.573 (q, 4H, J=7 Hz), 3.456 (br, s, 2H)6.513 (m, 2H), 6.970 (d, 1H, J=7.8 Hz). (20): 1.215 (t, 6H, J=7 Hz),2.617 (q, 4H, J=7 Hz), 6.570 (d, 1H, J=7.5 Hi), 6.650 (d, 1H, J=7.5 Hz),6.973 (m, 1H).

4,5-Diethyl-2-nitrotrifluoroacetylanilide (21) and 3,4-Diethyl-2nitrotrifluoroacetylanilide (22).

Into a 15-mL, single-necked, round-bottomed flask equipped with amagnetic stirrer, reflux condenser, and drying tube were added3,4-diethylaniline 19 (0.822 g, 5.52 mmol), ammonium nitrate (0.456 g,5.70 mmol; Baker), and trifluoroacetic anhydride (5 mL, 35.0 mmol;Sigma). To this mixture was added chloroform (10 mL) at 0° C. under N₂with stirring. The mixture was allowed to stir at room temperature for2.5 h. The mixture was poured into ice (20 g), extracted with chloroform(3×20 mL), and dried over sodium sulfate. The solvent was removed byaspirator to give a residue, which was separated by preparative TLC(Hexanes: EtOAc=8:2), giving 4,5-diethyl-2-nitrofluoroacetylanilide (21)(0.898 g, 56%) as a yellow solid; mp 58-60° C.; and3,4-diethyl-2-nitrotrifluoroacetylanilide (22) (0.30 g, 18%) as a brownsolid, mp 70-72° C. ¹H NMR (CDCl₃,300 MHz): δ (21): 1.275 (m, 6H), 2.742(m, 4H), 8.099 (s, 1H), 8.539 (s, 1H), 11.375 (s, 1H); (22): 1.262 (m,6H), 2.661 (m, 4H), 7.420 (d, 1H, J=8.7 Hz), 7.915 (d, 1H, J=8.7 Hz),8.742 (s, 1H).

4,5-Diethyl-2-nitroaniline (23).

To a solution of 4,5-diethyl-2-nitrotrifluoroacetylanilide (21) (0.898g, 3.1 mmol) in absolute alcohol (30 mL) was added 10% sodium hydroxideaqueous solution (10 mL). The solution was stirred at 80° C. (oil bath)for 4 h. Ethanol was evaporated in vacuo to leave an orange solid, whichwas collected by suction filtration, washed with water (3×15 mL), anddried in vacuo, giving 567 mg (95%) of the title compound 23 as anorange solid; mp 63-65° C. (lit. mp 64-65° C.; Lambooy, J. P., J. Am.Chem. Soc. 71:3756 (1949)); ¹H NMR (CDCl₃, 300 MHz) b 1.242 (m, 6H),2.568 (m, 4H), 5.846 (br, s, 2H), 6.593 (s, 1H), 7.899 (s, 1H).

4,5-Diethyl-1,2-diaminobenzene (24).

4,5-Diethyl-2-nitroaniline (23) (567 mg, 2.92 mmol) was dissolved inethyl acetate (20 mL). To this solution was added 10% Pd-C (142 mg, 20%;Aldrich). The mixture was agitated at room temperature under a pressureof 35 psi (H₂) for 5 h. The catalyst was removed through a column ofCelite (5 g) and washed with ethyl acetate (3×20 mL). The filtrates werecombined and evaporated in vacuo to give 4,5-diethyl-1,2-diaminobenzene24 (459 mg, 96%) as a colorless solid; mp 114-116° C. (lit. mp 114-115°C.; Lambooy, J. P., J. Am. Chem. Soc. 71:3756 (1949)); ¹H NMR (CDCl₃,300 MHz) δ 1.158 (t, 6H, J=7.5 Hz), 2.498 (q, 4H, J=7.5 Hz), 2.908 (br,s, 4H), 6.555 (s, 2H).

6,7-Diethyl-1,4-dihydroquinoxaline-2,3-dione(25).

4,5-Diethyl-1,2-diaminobenzene 24 (459 mg, 2.80 mmol) was dissolved in 4N hydrochloric acid (12 mL) at 90° C. Oxalic acid dihydrate (983 mg,6.47 mmoL) was added to this solution in one portion with stirring underN₂. The mixture was refluxed at 130-5° C. (oil bath) for 3 h., A yellowsolid precipitated, which was collected by suction filtration and driedin vacuo overnight, giving 538 mg (88%) of the title compound 25 as abrown solid; mp >360° C.; ¹H NMR (DMSO-d₆, 300 MHz) δ 1.092 (t, 6H,J=7.5 Hz), 2.510 (q, 4H, J=7.5 Hz), 6.867 (s, 2H), 11.742 (s, 2H). EIMSm/e 218 (95, M⁺), 203 (100, M⁺—CH₃). (HPLC purity 100%)

5-Nitro-6,7-diethyl-1,4-dihydroquinoxaline-2,3-dione (26).

6,7-Diethyl-2,3-quinoxalinedione 25 (218 mg, 1.0 mmol) was added totrifluoroacetic acid (8 mL; Sigma). To this suspension was addedpotassium nitrate (121.2 mg, 1.2 mmol; Baker) in one portion withstirring under N₂. The reaction was stirred at room temperature for 48h. Trifluoroacetic acid was evaporated in vacuo to give a residue. Water(10 mL) was added to this residue with vigorous stirring. A yellow solidprecipitated, which was collected by suction filtration and dried invacuo to give 182 mg (69%) of the title compound 26 as a yellow solid;mp 270-272° C. (dec.); ¹H NMR (DMSO-d₆,300 MHz) δ 1.137 (m, 6H), 2.622(m, 4H), 7.088 (s, 1H), 11.762 (s, 1H), 12.028 (s, 1H). EIMS m/e 263(100, M⁺). (HPLC purity 100%).

Example 36

Preparation of 5-Nitrocyclopento[g]-1,4-dihydroquinoxaline-2,3-dione

5-Acetamidoindan.

To a solution of 5.2 g (39 mmol) of 5-aminoindan in 15 mL of dioxanekept in an ice-bath was added dropwise 8 mL (8.6 g, 84 mmol) of aceticanhydride. The solution was stirred at room temperature for 16 h. It wasdiluted with 70 mL of water. The mixture was filtered, washed withwater, and dried to leave a gray solid 6.21 g (91%). ¹H NMR (CDCl₃),2.069 (m, 2), 2.165 (s, 3), 2.879 (m, 4), 7.20 (mb, 1), 7.145 (s, 2),7.441 (s, 1).

5-Acetamido-6-nitroindan.

To a solution of 5.59 g (31.9 mmol) of 5-acetamidoindan in 55 mL ofH₂SO₄ kept in an ice-bath was added portionwise 3.62 g of KNO₃ (35.8mmol). The solution was stirred in the ice-bath for 2 h and at roomtemperature overnight. It was added to 500 mL of ice-water and stirredfor 1 h. The precipitate was filtered, washed with water, and dried toleave a black solid, which was separated by chromatography (silica gel,eluted with hexane/ethyl acetate=10:1) to give 1.06 g (15%) of a yellowsolid. ¹H NMR (CDCl₃), 2.142 (m, 2), 2.279 (s, 3), 3.00 (m, 4), 8.44 (s,1), 8.575 (s, 1), 10.389 (s, 1).

5-Amino-6-nitroindan.

A mixture of 259 mg (1.16 mmol) of 5-acetamido-6-nitroindan in 4 mL of 2N HCl was heated at 85° C. for 9 h and cooled to room temperature. Thesolid precipitate was filtered, washed with water, and dried to leave201 mg (97%) of a crystalline yellow solid. ¹H NMR (CDCl₃), 2.068 (m,2), 2.843 (m, 4), 6.00 (sb, 2), 6.650 (s, 1), 7.942 (s, 1).

5,6-Diaminoindan.

A solution of 200 mg (1.12 mmol) of 5-amino 6-nitroindan and 1.14 g(6.01 mmol) of SnCl₂ in 8 mL of ethanol was heated at 70° C. for 2 h. Itwas evaporated to remove the ethanol. The residue was treated with 40%aqueous NaOH to pH =12. The mixture was diluted with 4 mL of water andextracted with CHCl₃(3×10 mL). The extract was dried (MgSO₄) andevaporated to leave a yellow crystalline solid (162 mg, 97%). ¹H NMR(CDCl₃), 2.012 (m, 2), 2.173 (t, 4, J=7.25), 3.301 (sb, 4), 6.614 (s,2).

Cyclopento[g]-1,4-dihydroquinoxaline-2,3-dione.

A mixture of 162 mg (1.09 mmol) of 5,6-diaminoindan and 108 mg (1.20mmol) of oxalic acid in 3 mL of 2N HCl was refluxed for 3 h and cooledto room temperature. The mixture was filtered, washed with water, anddried to leave an almost colorless solid (187 mg, 85%); mp >360° C.; ¹HNMR (DMSO-d₆), 1.985 (m, 2), 2.808 (t, 4, J=7.28), 6.957 (s, 2), 11.833(s, 2). MS, 202 (M^(+,) 100), 173 (90), 145 (10), 130 (20). HRMS, Calc.for CH₁₁N₁₀N₂O₂ 202.0738, found 202.0747.

5-Nitrocyclopento[g]-1,4-dihydroquinoxaline-2,3-dione.

To a stirred mixture of 84 mg (0.41 mmol) ofcyclopentot[g]-1,4-dihydroquinoxaline-2,3-dione in 3 mL of CF₃CO₂H wasadded in one portion 44 mg (0.43 mmol) of KNO₃. The mixture was stirredat room temperature for 14 h and the resulting red solution wasevaporated. The residue was diluted with water (4 mL). The mixture wasfiltered, washed with water, and dried to leave a yellow solid (74 mg,73%). ¹H NMR (DMSO-d₆), 2.040 (m, 2), 2.926 (t, 2, J=7.5), 3.085 (t, 2,J=7.5), 7.259 (s, 1), 11.175 (s, 1), 12.233 (s, 1).

Example 37

Preparation of7-Fluoro-6-(n-butoxy)-5-nitro-1,4-dihydroquinoxaline-2,3-dione (28)

To a mixture of 25 mg (1.0 mmol) of NaH and 50 mg (0.67 mmol) ofn-butanol was added 1.5 mL of DMSO and the mixture was stirred for 1 h.To the resulting solution was added 27 mg (0.11 mmol) of6,7-difluoro-5-nitroquinoxaline-2,3-dione (27) and the solution wasstirred at room temperature for 5 days. The solution was diluted with 3mL of water and acidified with 2 N HCl to pH=1. The precipitate wasfiltered, washed with water, and dried to leave 11 mg (33%) of a yellowsolid; mp 290-292° C.; ¹H NMR (DMSO-d₆, 0.882 (t, 3, J=7.4), 1.360 (m,2), 1.595 (m, 2), 4.084 (t, 2, J=6.3), 7.151 (1, d, J=11.54), 11.98 (m,1), 12.12 (m, 1), MS, 297 (M+, 20), 241 (100), 195 (20). HRMS, Calcd forC₁₂H₁₂FN₃O₅ 297.0755; found 297.0757.

Example 38

Preparation of7-Fluoro-6(3-phenylpropoxy)-5-nitro-1,4-dihydroquinoxaline-2,3-dione(29)

Compound 29 was prepared in a manner similar to that of compound 28.From 25 mg (1.0 mmol) of NaH, 61 mg (0.44 mmol) of 3-phenylpropanol and28 mg (0.11 mmol) of 6,7-difluoro-5-nitroquinoxaline-2,3-dione (27) in 2mL of DMSO was obtained 25 mg (63%) of a yellow solid; mp 280-282° C.;¹H NMR (DMSO-d₆), 1.923 (m, 2), 2.653 (t, 2, J=7.8), 4.103 (t, 2,J=6.1), 7.135-7.306 (m, 6), 11.98 (m, 1), 12.131 (s, 1). ¹⁹F NMR,−133.87 (mb). MS, 359 (M+, 20), 243 (15), 119 (40), 91 (100). NRMS,Calcd for C₁₇H₁₄FN₃O₅ 359.0911; found 359.0927.

Example 39

Preparation of 6-Chloro-7-ethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione(37)

3-Ethyl-5-nitroanaline (30).

To 160 mL of concentrated H₂SO₄ stirred at room temperature was added24.5 g (0.20 mol) of 2-ethylaniline. The resulting solution was kept inan acetone-ice bath (−10 to −5° C.) and 10.5 mL (15.75 g, 0.225 mol) offuming HNO₃ (d 150, >90% HNO₃) in 20 mL of concentrated H₂SO₄ was addeddropwise. The solution was stirred in the acetone-ice bath for 30 minafter addition of HNO₃ and then allowed to warm to room temperature. Itwas poured into 1,000 mL of ice, whereupon the mixture was neutralizedby ammonium hydroxide (30%) to pH=8. The mixture was stirred for 1 h,filtered, washed with water, and dried to leave 32.2 g (97%) of redsolid. Recrystallization with 100 mL of absolute alcohol yielded 25.8 gof a yellow solid; mp 60-61° C. (Lambooy & Lambooy, J. Med. Chem.16:765-770 (1973): 63-64° C.); ¹H NMR (CDCl₃), 1.128 (t, 3, J=7.5),2.565 (q, 2, J=7.6), 3.915. (sb, 2), 7.177 (d, 1, J=8.2), 7.502 (d, 1,J=2.2), 7.582 (dd, 1, J=2.2, 8.3).

3-Chloro-4-ethylnitrobenzene (31).

A mixture of 8.3 g (50 mmol) of 30 in 40 mL of concentrated HCl and 40mL of H₂O was heated at 80° C. for 1 h. The mixture was cooled in an icebath and a solution of 3.6 g (52 mmol) of NaNO₂ in 6 mL of H₂O was addeddropwise. The solution was stirred in an ice bath for I h after additionof NaNO₂. The solution was added dropwise to a stirred solution of 10 g(102 mmol) of CuCl in 40 mL of concentrated HCl kept in an ice bath. Themixture was stirred in the ice bath for 1 h after addition of thediazonium solution and extracted with CHCl₃ (3×40 mL). The extract wasdried (MgSO₄) and evaporated to leave 9.2 g (99%) of pale-red oil. ¹HNMR (CDCl₃), 1.276 (t, 3, J=7.5), 2.851 (q, 2, J=7.5), 7.408 (d, 1,J=8.5), 8.064 (dd, 1, J=2.0, 8.4), 8.227 (d, 1, J=2.0).

3-Chloro-4-ethylaniline (32).

A solution of 8.9 g (48 mmol) of 31 and 54.6 g (241 mmol) of SnCl₂, 2H₂Oin 100 mL of absolute alcohol was refluxed for 1 h. It was evaporated toremove most of the solvent and the residue was treated with 2N NaOH topH =9. The mixture was filtered and the solid was washed with methanol(10 mL) and ethyl acetate (200 mL). The filtrate was separated and theaqueous phase was extracted with ethyl acetate (2×50 mL). The combinedorganic solution was dried (MgSO₄) and evaporated to leave 7.31 g (98%)of liquid. ¹H NMR (CDCl₃), 1.178 (t, 3, J=7.5), 2.639 (q, 2, J=7.4),6.532 (dd, 1, J=2.2, 8.1), 6.700 (d, 1, J=2.2), 6.996 (d, 1, J=8.2).

4-Chloro-5-ethyl-2-trifluoracetamidonitrobenzene (33).

To 25 mL of (CF₃CO)₂O kept in an ice bath was added dropwise 2.56 g(16.4 mL) of 32 and the resulting mixture was stirred at roomtemperature for 1 h. To the mixture, kept in the ice bath, was added inportion 1.72 g (17.0 mmol) of KNO₃, and the resulting solution wasstirred in the ice bath for 1 h and at room temperature overnight. Thesolution was added to 100 mL of ice/water yielding a precipitate thatwas filtered, washed with water, and dried to give a pale-yellow solid.It was crystallized with absolute alcohol (30 mL) to give 2.55 g (52%)of a colorless solid; mp 82-83° C.; ¹H NMR (CDCl₃), 1.293 (t, 3, J=7.5),2.825 (q, 2, J=7.7), 8.197 (s, 1), 8.805 (s, 1), 11.35 (mb, 1).

3-Chloro-4-ethyl-6-nitroaniline (34).

A mixture of 1.4 g (4.7 mmol) of 33 and 10 mL of 7% K₂CO₃ inmethanol/H₂O (3:2) was stirred at room temperature for 2 h. It wasdiluted with 15 mL of water, filtered, washed with water, and dried toleave 691 mg (73%) of a yellow solid; mp 100-101° C.; (Lit. 104-106° C.Lambooy & Lambooy, supra). ¹H NMR (CDCl₃), 1.228 (t, 3, J=7.5), 2.665(q, 2, J=7.5), 5.957 (sb, 2), 6.859 (s, 1), 7.995 (s, 1).

Ethyl-N-(3-chloro-4-ethyl-6-nitrophenyl) Glycinate (35).

A mixture of 200 mg (1.0 mmol) of 34, 140 mg of K₂CO₃, and 2 mL of ethylbromoacetate was heated at 130° C. for 3 days. The mixture was cooled toroom temperature, diluted with 10 mL of 1 N NaOH, and stirred at roomtemperature for 4 h. The mixture was acidified with 2 N HCl to pH=1,filtered, washed with water, and dried to leave 280 mg (97%) of a yellowsolid. ¹H NMR (DMSO-d₆), 1.144 (t, 3, J=7.4), 1.213 (t, 3, J=7.1), 2.620(q, 2, J=7.4), 4.165 (q, 2, J=7.1), 4.271 (d, 2, J=5.8), 7.042 (s, 1),8.034 (s, 1), 8.281 (s, 1).

6-Chloro-7-ethyl-3,4-dihydroquinoxaline-2-one (36).

A solution of 280 mg (0.977 mmol) of 35, 950 mg (4.21 mmol) ofSnCl₂.2H₂O, and 8 mL S of absolute alcohol was refluxed for 4 h andcooled to room temperature. It was evaporated and the residue wastreated with 1 N NaOH to pH=10. The mixture was filtered, washed withwater, and dried to leave a solid. The solid was stirred with 15 mL ofethyl acetate and filtered. The filtrate was evaporated to leave 137 mg(66%) of solid. ¹H NMR (DMSO-d₆), 1.080 (t, 3, J=7.4), 2.503 (q, 2,J=7.2), 3.700 (s, 2), 6.036 (s, 1), 6.632 (s, 1), 6.662 (s, 1), 10.281(s, 1).

6-Chloro-7-ethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione(37).

To a solution of 137 mg (0.65 mmol) of 36 in 4 mL of CF₃CO₂H kept in anice bath was added dropwise 0.4 mL of fuming HNO₃. The solution wasstirred in the ice bath for 1 h and at room temperature overnight. Itwas evaporated and the residue was treated with 6 mL of water andstirred for 10 min. The mixture was filtered, washed with water, anddried to leave a yellow solid, 140 mg (80%); mp >330° C.; ¹H NMR(DMSO-d₆), 1.161 (t, 3, J=7.4), 2.712 (q, 2, J=7.6), 7.192 (s, 1),12.209 (mb, 2). MS, 269 (M+, 100), 254 (12), 226 (15), 160 (20). HRMS,Cacld for C₁₀H₈ClN₃O₄ 269.0198; Found 269.0196.

Example 40

Preparation of 6-Chloro-7-ethyl-1,4-dihydroquinoxaline-2,3-dione (39)

1,2-Diamine-4-chloro-5-ethylbenzene (38). A solution of 271 mg (1.35mmol) of 34 and 1.24 g (5.49 mmol) of SnCl₂.2H₂O, in 5 mL of absolutealcohol was refluxed for 2 h. It was evaporated and the residue wastreated with 1 N NaOH to pH=10. The mixture was extracted with CH₂Cl₂(3×10 mL). The extract was dried (MgSO₄) and evaporated to leave 229 mg(99%) of a yellow solid. ¹H NMR (CDCl₃), 1.165 (t, 3, J=7.5), 2.594 (q,2, J=7.2), 6.560 (s, 1), 6.696 (s, 1).

6-Chloro-7-ethyl-1,4-dihydroquinoxaline-2,3-dione(39).

A mixture of 228 mg (1.33 mmol) of 38 and 124 mg (1.38 mmol) of oxalicacid in 4 mL of aqueous 2N HCl was refluxed for 4 h. It was cooled toroom temperature, filtered, and dried to leave 275 mg (92%) of a brownsolid; mp >400° C.; ¹H NMR (DMSO-d₆), 1.134 (t, 3, J=7.4), 2.635 (q, 2,J=7.4), 7.026 (s, 1), 7.107 (s, 1), 11.890 (s, 1), 11.927 (s, 1). MS,224 (M⁺, 100), 209 (45), 181 (80). HRMS, Cacld for C₁₀H₉ClN₂O₂ 224.0348;Found 224.0359.

Example 41

Preparation of 6-Chloro-7-ethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione(37) and 7-Chloro-6-ethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione (40)

To a mixture of 232 mg (103 mmol) of 39 in 6 mL of CF₃CO₂H kept in anice bath was added 113 mg of KNO₃ portionwise. The mixture was stirredin the ice bath for 1 h and at room temperature overnight. To the Lsolution was added 28 mg of KNO₃ and it was stirred overnight. It wasevaporated and the residue was treated with 10 mL of water, filtered,washed with water, and dried to leave 227 mg (81%) of a yellow solid. ¹HNMR (DMSO-d₆), (37): 1.14 (m, 3), 2.712 (q, 2, J=7.4), 7.193 (s, 1),12.209 (mb, 2), and (40): 1.14 (m, 3), 2.582 (q, 2, J=7.4), 7.303 (s,1), 12.0 (mb, 1), 12.17 (mb, 1). 37:40=1:1.

Example 42

Preparation of6-Chloro-7-Fluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione(41)

A mixture of 25 mg (0.095 mmol) of6-amino-7-fluoro-5-trifluoromethylquinoxaline-2,3-dione (29) in 1.0 mLof concentrated HCl was stirred in an ice-bath for 1 h. To the resultingsolution was added dropwise a solution of 40 mg (0.58 mmol) of NaNO₂ in0.1 ml of H₂O and the solution was stirred in an ice-bath for 4 h. Tothe solution was added dropwise a solution of 60 mg of CuCl in 0.3 mL of6 N HCl. The resulting mixture was stirred in an ice-bath for 4 h and atroom temperature overnight. The mixture was diluted with 1 mL of H₂O andstirred for 10 min. followed by another 1 mL of H₂O with stirring foranother 10 min. It was filtered, washed with water, and dried to leave21 mg (78%) of a pale-yellow solid; mp 323-325° C.; ¹H NMR (DMSO-d₆),7.337 (d, 1, J=9.34), 10.95 (mb, 1), 12.309 (s, 1). MS, 282 (M⁺, 100),254 (20), 234 (80), 207 (40). HRMS, Cacld for C₉H₃ClF₄N₂O₂ 281.9816;Found 281.9833.

4,5-Dimethoxy-1,2-dinitrobenzene (43).

The procedure of Wulfman and Cooper, Synthesis 1978&924 was adopted forthis reaction. 1,2-Dimethoxybenzene 42 (20.8 g, 0. 15 mol; Aldrich) wasadded dropwise over 30 min. to vigorously stirred 70% nitric acid (175mL) under nitrogen. The temperature was kept below 50° C. during theaddition. Shortly after the addition was complete, a yellow solidprecipitated. The mixture was then heated to 70-80° C. for about 2 h(until evolution of NO₂ ceased). The mixture was allowed to cool below40° C. and was then poured into ice/water (1,000 mL) and filtered bysuction filtration. The yellow solid was slurried in saturated sodiumbicarbonate (500 ml) overnight. The crude product was isolated bysuction filtration and then purified by recrystallization from ethylalcohol (1,000 mL). The tide compound was obtained as fine yellowneedles (23 g, 67%); mp 128-130° C. (lit. mp 127-128° C.); ¹H NMR(CDCl₃,300 MHz) δ 4.016 (s, 6H), 7.337 (s, 2H).

6,7-Dimethoxy-2,3-quinoxalinedione (45) (from 43, two steps).

4-5-Dimethoxy-1,2-dinitrobenzene (43) (912 mg, 4.00 mmol) was dissolvedin ethyl acetate (30 mL). To this solution was added 10% Pd/C (228 mg,20%; Aldrich). The mixture was then stirred at room temperature under apressure of 40 psi (H₂) for 14 hr. The catalyst was removed through acolumn of Celite (5 g) and washed with ethyl acetate (3×15 mL) undernitrogen. The extracts were combined and the solvent was removed to givethe diamine (44) as a nearly colorless solid. The ¹H NMR spectrum wasconsistent with the assigned structure. Diamine (44) was dissolved in 4N hydrochloric acid (7 mL), and oxalic acid dihydrate (504 mg, 4.00mmol; Fisher) was added to this solution in one portion with stirringunder N₂. The mixture was refluxed at 130-5° C. (oil bath) for 3 hr. Ayellow solid precipitated, which was collected by suction filtration anddried in vacuo overnight, yielding 725 mg (82% based on compound 43) ofthe title compound as a pale yellow solid; mp 345-346° C.; ¹H NMR(DMSOd₆, 300 MHz) a 3.677 (s, 3H), 6.681 (s, 2H), 11.695 (s, 2H). EIMSm/e 222 (100, M⁺). (HPLC purity 99%).

6,7-Dihydroxy-2,3-quinoxalinedione (46).

To a suspension of 6,7-dimethoxy-2,3-quinoxalinedione (45) (222 mg, 1.0mmol) in 2 mL of methylene dichloride was added 5 mL of a solution ofboron tribromide in methylene dichloride (1 M, Aldrich). The resultingmixture was stirred at room temperature for 24 hr. The mixture was thenpoured into ice-water (10 g) to form a suspension. Aquous sodiumhydroxide (20%, 10 mL) was added to the suspension to form a redsolution. The solution was acidified with 6 N HCl (10 mL) to pH =1. Thesuspension was centrifuged, washed with methanol, and dried in vacuo,giving 170 mg (88%) of the product as a brown solid; mp >350° C.; ¹H NMR(DMSO-d₆, 300 MHz) b 6.541 (s, 2H), 8.989 (s, 2H), 11.543 (s, 2H). EIMSm/e 194 (100, M⁺). (HPLC>99%).

5-Acetoxy-6,7-dimethoxy-2,3-quinoxalinedione (47).

6,7-Dimethoxy-2,3-quinoxalinedione (45) (222 mg, 1.0 mmol) was dissolvedin glacial acetic acid (10 mL) at 100° C. (oil bath). The oil bath wasremoved and fuming nitric acid (53 μL, 1.2 mmol; Baker) was addeddropwise to the solution. The reaction mixture was then stirred at roomtemperature for 24 h. The mixture was filtered and washed with water togive a white solid, giving 150 mg (54%) of the title compound, mp321-322° C.; ¹H NMR (DMSO-d₆, 300 MHz) δ 2.300 (s, 3H), 3.619 (s, 3H),3.746 (s, 3H), 6.661 (s, 1H), 11.700 (s, 1H), 11.832 (s, 1H). EIMS m/e280 (M⁺, 30), 238 (100). Anal. Calcd for C₁₂H₁₂N₂O₆: C, 51.43; H, 4.32;N, 10.00. Found: C, 51.29; H, 4.04; N, 9.90. (HPLC purity >99%).

5-Hydroxy-6,7-dimethoxy-2,3-quinoxalinedione (48).

5-Acetyloxy-6,7-dimethoxy-2,3-quinoxalinedione (47) (30 mg, 0.107 mmol)was added to a single-necked 15-mL flask. Sodium hydroxide aqueoussolution (2 N, 1 mL) was added to the flask under nitrogen withstirring. The solution was then stirred at room temperature for 24 h.The solution was diluted with water (3 mL) and then acidified with HCl(4 N, 1 mL) to pH=2 to give a brown solid, which was collected byfiltration and dried in vacuo, giving 23 mg (86%) of the title compound;mp 285-286° C. (dec); ¹H NMR (DMSO-d₆, 300 MHz) a 3.688 (s, 3H), 3.694(s, 3H), 6.227 (s, 1H), 9.769 (s, 1H), 11.016 (s, 1H), 11.658 (s, 1H).EIMS m/e 238 (100, M⁺), HPLC purity >96%.

5,6,7-Trihydroxy-2,3-quinoxalinedione (49).

To a suspension of 5-hydroxy-6,7-dimethoxy-2,3-quinoxalinedione (48) (50mg, 0.22 mmol) in 2 mL of methylene dichloride was added 2 mL of asolution of boron tribomide in methylene dichloride (1 M, Aldrich). Theresulting mixture was stirred at room temperature for 12 hr. The mixturewas poured into ice-water (5 g) to form a suspension. Aqueous sodiumhydroxide (20%, 2 mL) was added to the suspension to form a redsolution. Then the solution was acidified with 6 N HCl (5 mL) to pH=1.The suspension was centrifuged, washed with methanol, and dried in vacuogiving 37 mg (80%) of the product as a brown solid; mp >350° C.; ¹H NMR(DMSO-d₆,300 MHz) δ 6.149 (s, 1H), 9.113 (s, 1H), 9.190 (s, 2H), 10.713(s, 1H), 11.501 (s, 1H) (HPLC >98%).

5(2-Phenylacetyloxy)-6,7-dimethoxy-2,3-quinoxalinedione (50).

To a suspension of 5-hydroxy-6,7-dimethoxy-2,3-quinoxalinedione (48) (50mg, 0.22 mmol) in 3 mL of methylene dichloride was added 0.14 mL oftriethylamine and phenylacetyl chloride (77.5 mg, 0.5 mmol, Aldrich) at0° C. The resulting mixture was stirred at room temperature for 12 hr.The reaction mixture was then poured into ice-water (5 g). A white solidprecipitated and was collected by suction filtration, washed with water(3×5 mL) and ethyl acetate (3×2 mL), and dried in vacuo, giving 60 mg(77%) of the product as a white solid; mp 298-300° C.; ¹H NMR (DMSO-d₆,300 MHz) δ 3.405 (s, 3 H), 3.728 (s, 3H), 4.024 (s, 2H), 6.653 (s, 1H),7.359 (m, 5H), 11.773 (s, 1H), 11.846 (s, 1H). EIMS m/e 356 (25, M⁺),238 (100). (HPLC 100%).

Example 44

4-Chloro-2,3-dinitrotoluene (52).

4-Chloro-2-nitrotoluene 51 (1.716 g, 10 mmol; Aldrich) was dissolved in10 mL of concentrated sulfuric acid. To this solution was addedpotassium nitrate (1.212 g, 12 mmol; Baker). The resulting mixture wasstirred at 80° C. for 12 hr. The mixture was poured into ice-water (20g). The precipitate was collected by suction filtration and dried invacuo, giving 2 g of a mixture of three isomers. After flashchromatography, 0.519 g of the desired 4-chloro-2,3-dinitrotoluene (52)(R_(f)=0.36 Hexanes: Ethyl acetate=7:3) was obtained in 24% yield; mp78-80° C.; ¹H NMR (CDCl₃,300 MHz) δ 2.475 (s, 3H), 7.445 (d, 1H, J=8.4Hz), 7.610 (d, 1H, J=8.4 Hz).

5-Chloro-8-methyl-2,3-quinoxalinedione (54) (from 52 two steps).

4-Chloro-2,3-dinitrotoluene (52) (519 mg, 2.4 mmol) was dissolved inethyl acetate (6 mL). To this solution was added 10% Pd/C (130 mg, 20%;Aldrich). The mixture was then stirred at room temperature under apressure of 60 psi (H₂) for 8 hr. The catalyst was removed through acolumn of Celite (5 g) and washed with ethyl acetate (3×15 mL) undernitrogen. The extracts were combined and the solvent was removed to givethe diamine 53 as a brown oil. The ¹H NMR spectrum was consistent withthe assigned structure. Diamine 53 was dissolved in 4 N hydrochloricacid (8 mL), and oxalic acid dihydrate (260 mg, 2.05 mmol; Fisher) wasadded to this solution in one portion with stirring under N₂. Themixture was refluxed at 130-5° C. (oil bath) for 6 hr. A brown solidcame out that was collected by suction filtration and dried in vacuoovernight, giving 320 mg (63% based on compound 53) of the titlecompound as a brown solid; mp >350° C.; ¹H NMR (DMSO-d₆, 300 MHz) δ2.288 (s, 3H), 6.915 (d, 1 H, J=8.4 Hz), 7.080 (d, 1 H, J=8.4 Hz),11.230 (s, 1H), 11.277 (s, 1H). EIMS m/e 210 (100, M⁺). (HPLC purity96%).

5-Chloro-6,7-dinitro-8-methyl-2,3-quinoxalinedione (55).

To the suspension of 5-chloro-8-methyl-2,3-quinoxalinedione (54) (180mg, 0.855 mmol) in trifluoroacetic acid (4 mL) was added potassiumnitrate (259 mg, 2.56 mmol). The resulting mixture was allowed to refluxfor 24 hr. Trifluoroacetic acid was evaporated in vacuo. Water (3 mL)was added and the solid was collected by suction filtration and dried invacuo, giving 200 mg (78%) of the product as a pale yellow solid; mp346-348° C.; ¹H NMR (DMSO-d₆, 300 MHz) δ 2.340 (s, 3H), 11.898 (s, 1H),12.070 (s, 1H). EIMS m/e 300 (20, M⁺, ³⁵Cl), 149 (100). (HPLC>97%).

Example 45

Preparation of6-Chloro-1,4-dihydro-7-fluoro-5-nitroquinoxaline-2,3-dione

1-Chloro-2,5-difluoro-4-nitrobenzene. To a stirred solution of1-chloro-2,5-difluorobenzene (0.770 g, 5.18 mmol) in concd H₂SO₄ (8.0mL) at 0° C., KNO₃.(0.525 g, 5.19 mmol) was added in one lot. Theresulting yellow solution was allowed to warm to room temperature andstirred overnight at room temperature. It was then poured into ice (80g) and extracted with ethyl acetate (75 mL). The ethyl acetate was driedover anhydrous Na₂SO₄, removed under vacuum and the residue was driedfurther under vacuum to afford 0.845 g (85%) of title compound as ayellow liquid; ¹H NMR (CDCl₃) δ 7.43 (dd, 1H, J₁=9.6 Hz, J₂=6.0 Hz),7.93 (t, 1H, J=7.2 Hz).

N-(5′-Chloro-4′-fluoro-2′-nitrophenyl)glycine sodium salt.

To a stirred solution of 1-chloro-2,5-difluoro-4-nitrobenzene (0.825 g,4.26 mmol) in ethanol (8.0 mL), was added a solution of sodium glycinate(0.415 g, 4.27 mmol) in water (1.5 mL). The resulting suspension wasrefluxed for 60 h. The solution was cooled to room temperature and theprecipitated bright orange solid was filtered, washed with cold ethanol(5 mL), and dried under vacuum to give 0.673 g (64%) title compound as abright orange powder; ¹H NMR (DMSO-d₆) δ 3.50 (d, 2H, J=3.6 Hz), 7.04(d, 1H, J=6.6 Hz), a 8.01 (d, 1H J=9.9 Hz), 8.74 (s, 1H). The unreacted1-chloro-2,5-difluoro-4-nitrobenzene was recovered almost quantitativelyfrom the filtrate.

6-Chloro-3,4-dihydro-7-fluoro-quinoxaline-2(1H)-one. A suspension ofN-(5′-chloro-4′-fluoro-2′-nitrophenyl)glycine sodium salt (0.650 g, 2.61mmol) and tin (11) chloride dihydrate (1.770 g, 7.845 mmol) in ethanol(10.5 mL) was refluxed for 1 h. It was then cooled to room temperatureand 5 mL ethanol was removed under vacuum. The precipitated solid wascollected by filtration under vacuum, washed with ethanol (3 mL) anddried to obtain 0.311 g (59%) title compound as an off-white powder; ¹HNMR (DMSO-d₆) δ 3.69 (s, 2H), 6.08 (s, 1H), 6.65 (d, 1H, J=9.9 Hz), 6.71(d, 1H, J=7.2 Hz), 10.39 (s, 1H). The solvent from the filtrate wasremoved under vacuum. The residue was diluted with water (15.0 mL) andthe pH was adjusted with 10% Na₂CO₃ to ˜9. The resulting suspension wasextracted with ethyl acetate (50 mL). The ethyl acetate was dried overanhydrous Na₂SO₄ and removed under vacuum to yield further 0.118 g (22%)of pure (¹H NMR) title compound for a combined yield of 81%.

6-Chloro-1,4-dihydro-7-fluoro-5-nitroquinoxaline-2,3-dione.

To a stirred suspension of6-chloro-3,4-dihydro-7-flouroquinoxaline-2(1H)-one (0.140 g, 0.698 mmol)in TFA (3.0 mL), excess fuming HNO₃ (0.20 mL) was added and theresulting red solution was stirred overnight at room temperature. Theresulting yellow suspension was poured into ice/water (15.0 mL). Theprecipitated solid was collected by vacuum filtration, washed with water(5.0 mL) and dried in a drying pistol (toluene reflux) to yield 0.124 g(69%) pure (HPLC) title compound as a yellow powder; ¹H NMR (DMSO-d₆) δ7.21 (d, 1H, J=9.3 Hz), 12.20 (s, 1H), 12.30 (s, 1H).

Example 46

Preparation of 7-Chloro-1,4-dihydro-6-ethyl-5-nitroquinoxaline-2,3-dione

2,5-Dichloro-1-ethylbenzene.

A suspension of mossy zinc (10 g) and mercuric chloride (0.5 g) in conedHCl (0.5 mL) and water (5 mL) was shaken for 5 min. The aqueous layerwas then decanted. To the residue was added concd HCl (7.5 mL) and water(7.5 mL) followed by 2′,5′-dichloroacetophenone (3.106 g, 16.43 mmol)and the suspension was refluxed for 4 h during which time, hourlyaddition of coned HCl (90.5 mL) was carried out. The resultingsuspension was cooled to room temperature and the aqueous layer wasdecanted. The residual solid was washed with ether (3×30 mL). Theaqueous layer was extracted with ether (3×50 mL). The combined etherlayer was washed with brine, dried over anhydrous Na₂SO₄, and removedunder vacuum. The residue was dried further under vacuum to obtain 2.664g crude product, which was purified on silica gel (Texane) to obtain0.813 g (28%) pure (¹H NMR) title compound as a colorless liquid; ¹H NMR(DMSO-d₆) 1.23 (t, 3H, J=7.5 Hz), 2.73 (q, 2H, J=7.5 Hz), 7.09-7.27 (m,3H).

2,5-Dichloro-4-ethyl-1-nitrobenzene.

To a stiffed solution of 2,5-dichloro-1-ethylbenzene (0.795 g, 4.68mmol) in coned H₂SO₄ (4.5 mL) at 0° C., KNO₃ (0.473 g, 4.68 mmol) wasadded in one portion. The resulting pale yellow solution was allowed towarm to room temperature and was stirred overnight at room temperature.It was then poured into ice (80 g) and extracted with ether (3×30 mL).The ether was dried over anhydrous Na₂SO₄, removed under vacuum, and theresulting oil was dried further under vacuum to obtain 0.943 g (92%)title compound as an oil; ¹H NMR (CDCl₃) δ 1.27 (t, 3H, J=7.5 Hz), 2.80(q, 2H, J=7.5 Hz), 7.42 (s, 1H), 7.94 (s, 1H).

N-(4′-Chloro-5′-ethyl-2′-nitrophenyl)glycine sodium salt.

To a solution of 2,5-dichloro-1-ethylbenzene (0.585 g, 2.66 mmol) inethanol (10.0 S mL) at room temperature was added a solution of sodiumglycinate (0.260 g, 2.68 mmol) in water (2.5 mL) and the resultingsuspension was refluxed for 2 days. The solution was then cooled to roomtemperature and the precipitated red solid was filtered, washed withethanol (4 mL), and dried under vacuum to give 0.086 g (13%) pure (¹HNMR) title compound as a red powder; ¹H NMR (DMSO-d₆) 1.15 (t, 3H, J=7.2Hz), 2.63 (q, 2H, J=7.2 Hz), 3.47 (d, 2H, J=3.0 Hz), 6.77 (s, 1H), 7.97(s, 1H), 8.77 (s, 1H). The unreacted 2,5-dichloro-4-ethyl-1-nitrobenzenewas recovered almost quantitatively from the filtrate.

7-Chloro-3,4-dihydro-6-ethylquinoxaline-2(1H)-one.

A suspension of N-(4′-Chloro-5′-ethyl-2′-nitrophenyl)glycine sodium salt(0.082 g, 0.31 mmol) and tin (II) chloride dihydrate (0.215 g, 0.953mmol) in ethanol (1.0 mL) was refluxed for 45 min. It was then cooled toroom temperature and the precipitated solid was filtered, washed withethanol (1.0 mL), and dried under vacuum to yield 0.048 g (72%) pure (¹HNMR) title compound as a light yellow powder; ¹H NMR (DMSO-d₆) δ 1.05(t, 3H, J=7.2 Hz), 2.52 (d, 2, J=7.2), 3.67 (s, 2H), 6.04 (s, 1H), 6.54(s, 1H), 6.67 (s, 1H), 10.25 (s, 1H).

7-Chloro-1,4-dihydro-6-ethyl-5-nitroquinoxaline-2,3-dione.

To a stirred suspension of7-chloro-3,4-dihydro-6-ethylquinoxaline-2(1H)-one (0.020 g, 0.095 mmol)in TFA (0.4 mL), excess fuming HNO₃ (0.04 mL) was added and theresulting red solution was stirred overnight at room temperature. Theresulting suspension was diluted with water (4 mL) and the precipitatedsolid was filtered, washed with water (2 mL), and dried under vacuum toyield 0.024 g (94%) pure (HPLC) title compound as a light yellow powder;¹H NMR (DMSO-d₆) δ 1.09 (t, 3H, J=7.2 Hz), 2.52 (q, 2H, J=7.2 Hz), 7.27(s, 1H), 11.99 (s, 1H), 12.15 (s, 1H).

Example 47

Preparation of5-Amino-7-chloro-1,4-dihydro-6-methylquinoxaline-2,3-dione

7-Chloro-1,4-dihydromethyl-5-nitroquinoxaline-2,3-dione.

To a stirred suspension of7-chloro-3,4-Dihydro-6-methyl-quinoxaline-2(1H)-one (0.100 g, 0.51 mmol)in TFA (3.0 mL) prepared as in Example 8, excess fuming HNO₃ (0.30 mL)was added and the resulting red solution was stirred overnight at roomtemperature. The solvent was removed under vacuum and the residuediluted with water (4.0 mL). The precipitated solid was filtered anddried under vacuum to yield 0.067 g (52%) pure (HPLC) title compound asa light yellow powder; mp-darkens at 340° C.; ¹H NMR (DMSO-d₆) δ 2.184(s, 3H), 7.263 (s, 1H), 11.948 (s, 1H), 12.144 (s, 1H); Anal forC₉H₆ClN₃O₄.H₂O calcd C, 40.85; H, 2.28; N, 15.87, found C, 40.63; H,2.05; N, 15.75.

5-Amino-7-chloro-1,4-dihydro-6-methylquinoxaline-2,3-dione.

A suspension of7-chloro-1,4-dihydro-6-methyl-5-nitroquinoxaline-2,3-dione (0.050 g,0.20 mmol) and tin (II) chloride dihydrate (0.133 g, 0.60 mmol) inethanol (1.0 mL) was refluxed for 6 h. The suspension was then cooled toroom temperature and the solid was collected by vacuum filtration,washed with ethanol (1 mL), and dried further under vacuum to obtain0.036 g (82%) pure (HPLC) title compound as an off-white solid;mp-darkens at 323° C.; 1H NMR (DMSO-d₆) δ 2.09 (s, 3H), 5.47 (s, 2H),6.46 (s, 1H), 11.15 (s, 11H, 11.72.(s, 1H).

Example 48

Preparation of7-Chloro-1,4-dihydro-6-ethylthio-5-nitroquinoxaline-2,3-dione

1-Chloro-2,4-difluoro-5-nitrobenzene.

To a stirred solution of 1-chloro-2,4-difluorobenzene (0.829 g, 5.58mmol) in concd H₂SO₄ (8.0 mL) at 0° C., KNO₃ (0.565 g, 5.59 mmol) wasadded in one portion. The resulting solution was allowed to warn to roomtemperature and stirred overnight at room temperature. It was thenpoured into ice (80 g) and extracted with ethyl acetate (75 mL). Theethyl acetate was dried over anhydrous Na₂SO₄, removed under vacuum, andthe resulting oil was dried further under vacuum to afford 1.007 g (93%)pure (¹H NMR) title compound as a light red oil; 1H NMR (CDCl₃) δ 7.17(dd, 1H, J₁=9.9 Hz, J₂=8.4 Hz), 8.24 (t, 1H, J=7.5 Hz).

N-(4′-Chloro-5′-fluoro-2′-nitrophenyl)glycine andN-(2′-chloro-5′-fluoro-4′-nitrophenyl)glycine sodium salt.

To a stirrd solution of 1-chloro-2,4-difluoro-5-nitrobenzene (1.000,5.167 mmol) in DMF (10.0 mL), was added dropwise, a solution of sodiumglycinate (0.502 g, 5.17 mmol) in water (2.0 mL). The solution wasstirred at 70° C. for 16 h. The resulting suspension was then cooled toroom temperature and the precipitated solid was filtered, washed withacetone (10 mL), and dried under vacuum to give 0.438 g (38%) red solidas a mixture of title compounds in a ratio of 3:1 (¹H NMR); 1¹H NMR(DMSO-d₆) δ 3.474 (d, 2H, J=4.5 Hz), 3.523 (d, 2H, J=3.9 Hz), 6.535 (d,1H, J=14.7 Hz), 6.871 (d, 1H, J=12.3 Hz), 6.976 (s, 1H), 8.068 (d, 1H,J=7.8 Hz), 8.168 (d, 1H, J=7.8 Hz), 8.867 (s, 1H). The separation of themixture was not feasible at this stage; hence, it was used as such forthe next reaction.

7-Chloro-3,4-dihydro-6-fluoroquinoxaline-2(1H)-one. A suspension ofN-(4′-chloro-5′-fluoro-2′-nitrophenyl)glycine sodium salt andN-(2′-chloro-5′-fluoro-4′-nitrophenyl)glycine sodium salt (0. 175 g,0.704 mmol) and tin (II) chloride dihydrate (0.475 g, 2.11 mmol) inethanol (3.5 mL) was refluxed for 30 min. It was then cooled to roomtemperature and the solvent was removed under vacuum. The residue wasdiluted with water (10 mL) and the pH was adjusted with saturated NaHCO₃(3.0 mL) to pH 8. The resulting white suspension was extracted withethyl acetate (30 mL). The ethyl acetate was dried over anhydrous Na₂SO₄and removed under vacuum to yield 0.041 g (29%) pure (¹H NMR) titlecompound as a light yellow powder; mp 217-219° C. (dec); ¹H NMR(DMSO-d₆) δ 3.75 (s, 2H), 6.37 (s, 1H), 6.59 (d, 1H, J=10.5 Hz), 6.74(d, 1H, J=7.2 Hz), 10.33 (s, 1H).

7-Chloro-1,4-dihydro-6-fluoro-5-nitroquinoxaline-2,3-dione.

To a stirred solution of7-chloro-3,4-dihydro-6-fluoro-quinoxaline-2(1H)-one (0.024 g, 0.12 mmol)in TFA (0.40 mL), excess fuming HNO₃ (0.020 mL) was added and theresulting red solution was stirred overnight at room temperature. Thesolvent was removed under vacuum and the residue was diluted with water(2.0 mL). The precipitated solid was filtered, washed with water (1.0mL), and dried in a drying pistol (toluene reflux) to yield 0.023 g(74%) pure (¹H NMR) title compound as a light yellow powder; mp 308-310°C.; ¹H NMR (DMSO-d₆) δ 7.37 (d, 1H, J=6.9 Hz), 12.02 (br s, 1H), 12.22(s, 1H); Anal for C₈H₃ClFN₃O₄.H₂O calcd C, 34.61; H, 1.09; N, 15.1,found C, 34.66; H, 1.09; N, 15.16%.

7-Chloro-1,4-dihydro-6-ethylthio-5-nitroquinoxaline-2,3-dione. Asolution of 7-Chloro-1,4-dihydro-6-fluoro-5-nitroquinoxaline-2,3-dione(0.100 g, 0.385 mmol) and ethanethiol, sodium salt (0.130 g, 1.55 mmol)in DMSO (1.0 mL) was stirred at room temperature for 24 h. The resultingsolution was diluted with water (5 mL) and acidified with concd HCl (4-5drops) to pH ˜5. The precipitated solid was collected by filtrationunder vacuum, washed with water (10 mL), and dried under vacuum toobtain 0.098 g (84%) pure (HPLC) title compound as a yellow powder; mpdarkens at 323° C.; ¹H NMR (DMSO-d₆) δ 1.04 (t, 3H, J=7.2 Hz), 2.82 (q,2H, J=7.5 Hz), 7.34 (s, 1H), 12.20 (s, 1H), 12.25 (s, 1H).

Example 49

Preparation of 7-Chloro-6-methyl-5-nitroquinoxaline-2(1H),3(4H)-dione

2-Chloro-5-fluoro-4-nitrotoluene.

To a stirred solution of 2-chloro-5-fluorotoluene (10.356 g, 71.628mmol, Lancaster, used as received) in conc. H₂SO₄ (70 mL) at 0° C., KNO₃(7.252 g, 71.72 mmol) was added in four equal portions. The resultingpale yellow solution was allowed to warm to room temperature and wasstirred overnight at room temperature. It was then poured into ice water(350 g) and extracted with ether (3×100 mL). Ether was dried overanhydrous Na₂SO₄, removed under vacuum, and the resulting oil was driedfurther under vacuum to afford 12.277 g (90%). of the title compound asan oil, which was used as such for the next reaction; ¹H NMR (CDCl₃); δ2.459 (s, 2H), 7.193 (d, 1H, J₁=11.1 Hz), 8.083 (d, 1H, J₁=6.6 Hz).

N₄(4′-Chlora-5′-methyl-2′-nitrophenyl)glycine potassium salt.

A suspension of 2-chloro-5-fluoro-4-nitrotoluene (11.200 g, 59.078mmol), glycine (4.500 g, 59.94 mmol), and KCO₃ (8.300 g, 60.05 mmol) inethanol (55 mL) and water (30 mL) was refluxed for 7 h. The resultingbright orange solid was cooled to room temperature. The solid wascollected by vacuum filtration, washed with water (50 mL), and dried invacuo. It was then taken up in acetate (150 mL), refluxed for 1 h, andcooled to room temperature. The orange solid was vacuum filtered anddried in vacuo to give 11.37 g (72%) pure (¹H NMR) title compound as ared powder; ¹H NMR (DMSO-d₆): δ 2.276 (s, 3H), 3.431 (d, 2H, J=4.2 Hz),6.848 (s, 1H), 7.963 (s, 1H), 8.773 (s, 1H).

7-Chloro-3,4-dihydro-6-methylquinoxaline-2(1H)-one.

To a stirred bright orange solution ofN-(4′-chloro-5′-methyl-2′-nitrophenyl)glycine potassium salt (0.097 g,0.36 mmol) in water (5.0 mL) at 80° C., sodium dithionite (0.500 g, 2.87mmol) was added in two equal portions. It instantly formed a whitesuspension, which was stirred at 80° C. for 1 h. It was then cooled atroom temperature and the solid was vacuum filtered, washed with water(5.0 mL), and dried in vacuo to yield 0.062 g (86%) of the titlecompound as a white powder; ¹H NMR (DMSO-d₆): δ 2.10 (s, 3H), 3.66 (s,2H, J=1.2 Hz), 5.98 (s, 1H), 6.53 (s, 1H), 6.67 (s, 1H), 10.20 (s, 1H).

7-Chloromethyl-5-nitroquinoxaline-2(1H),3(4H)-dione.

To a stirred suspension of7-chloro-3,4-dihydro-6-methyl-quinoxaline-2(1H)-one (0.040 g, 020 mmol)in TFA (0.50 mL), excess fuming H₂SO₃ (0.040 mL) was added and theresulting yellow suspension was stirred overnight at room temperature.It was poured into ice water (3 mL) and the precipitated solid wasvacuum filtered, washed with water (5 mL), and dried in vacuo to yield0.048 g (92%) pure (¹H NMR) of the title compound as a light yellowpowder; ¹H NMR (DMSO-d₆): δ 2.184 (s, 3H), 7.263 (s, 1H), 11.948 (s,1H), 12.144 (s, 1H).

Example 50

Evaluation of the Antinociceptive Effect of5-nitro-6,7-dimethylquinoxalinedione (NDMQX) in the Formalin Test inSwiss Webster Mice

Introduction

The co-existence of glutamate with substance P in dorsal root ganglionneurons (Battaglia & Rustioni, J. Comp. Neurol. 277:302-312 (1988)) andthe involvement of the N-methyl-D-aspartate (NMDA) receptor in “wind up”phenomenon in the dorsal horn nociceptive neurons (Davies & Lodge, BrainRes. 424:402-406 (1987); Dickenson & Sullivan, Brain Res. 506:31-39(1990); Haley, J. E. et al., Brain Res. 518:218-226 (1990)) suggest thatexcitatory amino acids are involved in nociceptive transmission. Thereis a great deal of evidence that demonstrates the analgesic effect ofthe NMDA receptor antagonists in different animal models of pain(Cahusac, P. M. et al., Neuropharmacol. 23:719-724 (1984); Murray, C. W.et al., Pain 44:179-185 (1991); Elliott, K. J. et al., Neurosci. Abs.17588 (1991); Nasstrom, J. et al., Eur. J. Pharmacol. 212:21-29 (1992);Coderre & Melzack, J. Neurosci. 12:3665-3670 (1992); Yamamoto & Yaksh,Anesthesiol. 77:757-763 (1992); Vaccarino, A. L. et al., Brain Res.615:331-334 (1993); Milan & Seguin, Eur. J. Pharmacol. 238:445447(1993)). However, the lack of access to the brain and/or the PCP-likeside effects are disadvantages to the use of some of these compounds.

It is well known that activation of the glycine modulatory siteassociated with the NMDA receptor is required for the activation of theNMDA receptor (Johnson & Ascher, Nature 325:529-531 (1987); Kleckner &Dingledine, Science 241:835-837 (1988); Thomson, A. M. et al., Nature338:422-424 (1989); Mayer, M. L. et al., Nature 338:425427 (1989)). Ithas been shown that 7-chloro-kynurenate (7CK), the antagonist for theglycine modulatory site coupled to NMDA receptor blocked the “wind up”phenomenon (Dickenson & Ayder, Neurosci. Let. 121:263-266 (1991)). Thus,another approach to produce analgesia would be to antagonize the glycinemodulatory site on the NMDA receptor.

Pain in clinical situations usually is prolonged and inflammatory innature. Thus, the use of animal models of persistent pain appears to bemore appropriate to evaluate the potential clinical use of novelanalgesic drugs. The present study was designed to evaluate theanalgesic effect of NDMQX, a novel and systemically active glycine/NMDAreceptor antagonist in the formalin-induced pain in Swiss Webster mice.

Methods

Subjects

Male Swiss Webster mice (25-35 g) obtained from Simonsen Laboratories,Inc. (Gilroy, Calif.) were used in all experiments. Mice were maintained4-6 per cage with free access to food and water under a 12/12 hrlight/dark cycle. Mice were housed for at least 5 days prior toexperimentation and used only once. All experiments were conductedduring the light cycle in a blind manner in which the observers were notinformed about the various treatments.

1. Effect of NDMQX in the Formalin Test

The formalin test performed was a modification of the method of Hunskaaret al. (Hunskaar, S. et al., J. Neurosci. Meth. 14:69-76 (1985)).Briefly, after a 60 minute accommodation period in plexiglass jars, micewere weighed and injected intraperitoneally (i.p.) with NDMQX (1-20mg/kg; N=6-10 mice/dose). Controls were injected with DMSO (1 mL/kg,i.p.). Thirty min later, mice were injected with formalin (20 μl of 5%solution) just under the skin of the right hind paw, transferred to theplexiglass jars, and immediately observed for licking or biting of theinjected paw for 1 hr. The amount of time that each mouse spent lickingthe injected paw was measured at 0-5 min (early phase) and 15-50 min(late phase).

Results

1. Effect of NDMQX in the Formalin Test

Systemic administration (i.p.) of NDMQX attenuated the mean time spentlicking in a dose-dependent manner in both phases of the formalin testin Swiss Webster mice (FIGS. 1 and 2, and Table V).

Conclusion

NDMQX produced a significant analgesic effect in both phases of theformalin-induced tonic pain in Swiss Webster mice demonstrating that thecompound has potential as an analgesic for conditions of tonic pain.

TABLE V Effect of NDMQX on the time spent licking in the formalin testEarly Phase (0.5 min) Late Phase (15-50 min) (mean ± s.e.m.) (mean ±s.e.m.) Treatment (sec per 5 min period) (sec per 5 min period) DMSO86.70 ± 8.65 33.00 ± 3.78 NDMQX (1.0 mg/kg) 72.57 ± 14.2 37.59 ± 5.37NDMQX (2.5 mg/kg) 78.50 ± 10.0 26.38 ± 5.43 NDMQX (5 mg/kg) 32.83 ± 8.2810.24 ± 2.66 NDMQX (10 mg/kg) 29.17 ± 14.9 10.19 ± 2.44 NDMQX (15 mg/kg) 6.83 ± 1.85  3.19 ± 1.37 NDMQX (20 mg/kg)  4.33 ± 2.85  0.05 ± 0.05

Example 51

The Anticonvulsant Effect and Antinociceptive Effect of5-nitro-6methyl-7-chloro-2,3-quinoxalinedione (NMCX) and5-nitro-6,7-dimethyl-2,3-quinoxalinedione (NDMQX) in the MES andFormalin Test.

The anti-convulsant and antinociceptive effect of NMCQX and NDMQX in theMES and in the formalin test in Swiss Webster mice were evaluated.

In the MES test, mice were injected i.p. with NMCQX (1.25-7.50 mg/kg) orNDMQX (5.00-15.00 mg/kg). Control mice were injected with Tris (0.05 M)or Arginine (0.1 M), respectively. Seizures were then induced byapplying a rectangular pulse, 50 mA, 60 pulses/sec, 0.8 msec pulsewidth, and 0.2 sec train length, at the time of the drugs' peak effect.Both compounds dose-dependently protected mice from generalizedclonic-tonic seizures induced by electroshock. Experimental results areshown in FIG. 36.

In the formalin test, following a 1 hr accommodation period, mice wereinjected i.p. with NMCQX (1.00-10.00 mg/kg, N=7-13 mice/dose) or NDMQX(0.5-20.00 mg/kg, N=8-9 mice/dose). Control mice were injected with Tris(0.5 M) or Bis-Tris (0.2 M), respectively. Mice were then injected withformalin. A s.c. injection of formalin produced a biphasic nociceptiveresponse, i.e., an early phase (0-5 min) followed by a tonic late phase(15-50 min). Both compounds produced a dose-dependent antinociceptiveeffect in both phases of the formalin test. The amount of time that eachmouse spent licking and/or biting the injected paw was recorded for 1hr.

The data suggest a modulatory role for the NMDA receptor in MES-inducedseizures and in formalin-induced nociception.

Example 52

Blockade of Morphine Tolerance by5-nitro-6,7-dimethyl-2,3-quinoxalinedione (NDMQX)

Introduction

Opioid analgesics are used for the management of pain. However, thedevelopment of tolerance to opioid analgesics impedes the therapeuticuse of these .drugs. Seeking alternative drugs to produce analgesiawithout development of tolerance or as an adjunct therapy to blocktolerance without interference with analgesia is an active area ofresearch. Tolerance develops after both acute and chronic morphineadministration (Kornetsly et al., Science 162:1011-1012 (1968); Way etal., J. Pharmacol. Exp. Ther. 167:1-8 (1969); Huidobro et al, J.Pharmacol. Exp. Ther. 198:318-329 (1976); Lutfy et al., J. Pharmacol.Exp. Ther. 256:575-580 (1991)). The results of recent studies havesuggested a modulatory role for N-methyl-D-aspartate (NMDA) receptor inmorphine tolerance (Trujillo et al., Science 251:85-87 (1991); Marek etal., Brain Res. 547:77-81 (1991); Tiseo et al., J. Pharmacol. Exp. Ther.264:1090-1096 (1993); Lutfy et al., Brain Res. 616:83-88 (1993).

Glycine is required for activation of the NMDA receptor (Johnson et al.,Nature 325:529-533 (1987); Kleckner et al., Science 241:835-837 (1988);Mayer et al., Nature 338:425-427 (1989); Thomson et al., Nature338:422-424 (1989)). Thus, another approach to block the NMDA receptor,and consequently block tolerance would be to antagonize the glycinemodulatory site of the NMDA receptor. This example shows the use of5-nitro-6,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione (NDMQX) to testthe hypothesis that inhibition at the NMDA receptor/glycine site mightbe a viable means of blocking morphine tolerance.

Materials and Methods

Subjects

Male Swiss Webster mice, weighing 25-35 g, obtained from SimonsenLaboratories, Inc. (Gilroy, Calif.) were used in all experiments. Micewere maintained 4-6 to a cage with free access to food and water under a12-hr light/12-hr dark cycle. Mice were housed for at least 5 days priorto experimentation and used only once. All experiments were conductedduring the light cycle in a blind manner in which the observers wereunaware of different treatments.

Formalin Test

A modification of a previously described method was used (Hunskaar etal., J. Neurosci. Meth. 14:69-76 (1985)). Briefly, mice were placed inPlexiglas jars for at least 1 hr for accommodation to the experimentalcondition. Formalin (20 μl of 5% formaldehyde solution in saline) wasinjected into the dorsal surface of the right hind paw using amicrosyringe (Hamilton Co., Reno, Nev.) with a 27-gauge needle. Micewere then transferred to the Plexiglas jars and immediately observed forlicking or biting of the injected paw for 1 hr. The amount of time thateach mouse spent licking or biting the injected paw was recorded forevery 5-min period for a 1 hr observation period.

1. Effect of NDMQX on Morphine Tolerance in the Formalin Test

Mice were injected daily for a period of 8 days with either vehicle(Bis-Tris; 0.2 M) or NDMQX (1, 20 and 40 mg/kg, i.p.; N=9-12mice/group). Mice were immediately, within a minute, injected witheither saline or morphine (20 mg/kg, s.c.). On day 9, mice were weighedand after a 1-hr accommodation period-injected with morphine (4 mg/kg,s.c.). Thirty min later, all mice were tested in the formalin test (seeabove).

2. Effect of NDMX on Morphine Antinociception in the Formalin Test

Mice were injected daily for a period of 8 days with either vehicle(Bis-Tris; 0.2 M) or NDMQX (40 mg/kg, i.p.; N=7 mice/group). One groupof mice (N =11) was left untreated.

On day 9, mice were weighed and after a 1-hr accommodation periodinjected with morphine (4 mg/kg,, s.c.). The untreated group was dividedinto two subgroups; half of which received saline while the other halfwas injected with morphine (4 mg/kg). Thirty min later, all mice weretested in the formalin test (see above).

Results

1. Effect of NDMQX on Morphine Tolerance in the Formalin Test

Morphine administration for 8 days produced tolerance in early and latephases of the formalin test (FIG. 7). A one-way ANOVA followed byNewman-Keul test revealed a statistically significant tolerance in micetreated chronically with morphine (F_(4,47)=4.20 and F_(4,47)=3.93 forthe early and late phases, respectively; p<0.05 or better). Tolerancedid not develop in mice treated with NDMQX followed by morphine. Theinhibitory effect of NDMQX on morphine tolerance was dose-dependent.NDMQX had no effect on morphine tolerance at 2 mg/kg; at higher doses iteither attenuated (at 20 mg/kg) or completely abolished (at 40 mg/kg)morphine tolerance (FIG. 7).

2. Effect of NDMQX on Morphine Antiociception in the Formalin Test

It was found that chronic administration of NDMQX for 8 days did notalter the antinociceptive effect of morphine in the formalin test (FIG.8). There was no significant difference in morphine-inducedantinociception between NDMQX-pretreated and control(vehicle-pretreated) groups (p>0.05). In addition, the level ofantinociception produced by morphine in these two groups was notsignificantly different from that of untreated (naive) mice.

Conclusion

NDMQX, a novel NMDA receptor/glycine site antagonist, blocked morphinetolerance in both phases of the formalin test. The blockade of toleranceby NDMQX was not due to potentiation of morphine-induced antinociceptionin the formalin test. These data suggest that the NMDA receptor isinvolved in modulation of morphine tolerance in an animal model of tonicpain. The blockade of morphine tolerance by NMDQX in the formalin testsuggests that antagonism at the glycine modulatory site associated withthe NMDA receptor is a viable means to inhibit NMDA receptor and blockmorphine tolerance. Therefore, the antagonism at the glycine modulatorysite associated with the NMDA receptor appears to be a potential sitefor the development of new drugs that could be used with morphine asadjunct therapy in the management of pain and prevention of tolerance.

Example 53

The Neuroprotective Effect of NDMQX in a Rat Model of Permanent FocalCerebral Ischemia Using Continuous Intravenous Drug Infusion

The N-methyl-D-aspartate (NMDA) subtype of excitatory amino acidreceptors appears to play a crucial role in neuronal degenerationinduced by focal cerebral ischemia. A number of competitive andnon-competitive NMDA receptor antagonists have been shown to providesignificant protection against neuronal degeneration in different animalmodels of focal ischemia (see Bullock et al., J. Neurotrauma 9. (Supp.2):S443-S462 (1992)). In this example, the neuroprotective effect of theNMDA receptor glycine site antagonist NDMQX in the rat model of thefocal ischemia is described.

Methods

1. Induction of Focal Ischemia

Male Sprague-Dawley rats (290-320 g) were incubated and maintained underanesthesia with ±2% of halothane. Body temperature was maintained at37.5° C. during surgery by means of a warming pad and a rectal probeconnected to the control unit. The common carotid arteries (CCA) wereisolated, and a loose silk ligature Was placed around each CCA. Avertical skin incision was made between the left orbit and the auditorycanal, posterior part of zygoma removed and small opening (2.0/2.5 mm)drilled dorsorostrally to the foramen ovale under constant salineirrigation. The dura was opened with a microsurgical hook and the braingently retracted with a fine spatula to expose the bifurcation of theinternal carotid artery and the middle cerebral artery. The ipsilateralCCA was ligated and the MCA coagulated from its origin to the olfactorytract Two hours after MCA occlusion, the clip from the contralateral CCAwas removed.

2. Drug Administration

NDMQX was dissolved in 0.1 M L-arginine and 5% glucose and infused as aslow 10 mg/kg i.v. bolus (immediately after MCA occlusion) followed by 7mg/kg/h infusion for 22 hours.

3. Histology

Brains were sliced into 2 mm blocks and stained with tetrazolium red(2,3,5-triphenyl tetrazolium chloride). The areas of brain damage wereassessed using an image analyzer (Image-1, Universal ImagingCorporation, PA) to determine the volume of cortical and subcorticalinfarction by integration of areas and the distance between each level.

Results

NDMQX produced a significant reduction (71%, F_(1,19)=11.57, p<0.05,ANOVA) in cortical infarct volume. See FIG. 9.

Conclusion

NDMQX provided significant neuroprotection in a rat model of focalischemia.

Example 54

Neuroprotective Effect of NMCQX in a Rat Model of Permanent FocalCerebral Ischemia Using Continuous Intravenous Drug Infusion

Example 52 was repeated, except that NMCQX was substituted for the NDMQXemployed therein. For the drug administration, the NMCQX was dissolvedin 0.05 Tris and 5% glucose. It was infused as a slow 2 mg/kg i.v. bolus(immediately after MCA occlusion) followed by 1.4 mg/kg/h infusion for22 hours.

NMCQX produced a significant reduction (75%, F_(1,18)=5.24, p<0.05,ANOVA) in cortical infarct volume. See FIG. 10.

Thus, NMCQX also provided significant neuroprotection in a rat model offocal ischemia.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations, and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents and publications cited herein are fullyincorporated by reference herein in their entirety.

What is claimed is:
 1. A method for the preparation of a1,4-dihydroquinoxaline-2,3-dione having the Formula:

or a tautomer thereof; wherein R¹ is nitro; R² is haloalkyl, halo,cyano, alkyl, or alkoxy; R³ is haloalkyl, halo, cyano, alkyl, or alkoxy;and R⁴ is hydrogen; comprising reaction of a compound having theFormula:

or a tautomer thereof; wherein R¹ is hydrogen; R² is haloalkyl, halo,cyano, alkyl, or alkoxy; R³ is haloalkyl, halo, cyano, alkyl, or alkoxy;and R⁴ is hydrogen; with fuming nitric acid; and isolating the1,4-dihydroquinoxaline-2,3-dione so produced.
 2. A method of treating orpreventing the adverse consequences of the hyperactivity of the NMDAreceptor, comprising administering to an animal in need of suchtreatment or prevention an effective amount of a compound having theFormula:

or a tautomer or a pharmaceutically acceptable salt thereof; wherein R¹is alkyl, azido, alkoxy, hydroxy, haloalkyl, halo, nitro, cyano oralkanoylamino; R² is alkyl, azido, alkoxy, aralkoxy, cyano, haloalkyl,halo, hydroxy or nitro; R³ is alkyl, azido, alkoxy, cyano, halo,haloalkyl or hydroxy; R⁴ is hydrogen or fluoro; provided that (a) atleast one of R¹, R² or R³ is hydroxy or azido, or (b) at least one of R²or R³ is alkyl or alkoxy, or (c) R¹ is cyano.
 3. The method of claim 2,wherein said compound is6,7-dimethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-fluoro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-chloro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-bromo-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione,6-chloro-7-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione,6,7-diethyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione,5-cyano-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione, or5-azido-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione.
 4. The method ofclaim 2, wherein said compound is7-chloro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione.
 5. Themethod of claim 2, wherein said compound is5-cyano-6,7-dichloro-1,4-dihydroquinoxaline-2,3-dione.
 6. The method ofclaim 2, wherein said compound is administered as an ammonium saltcomprising said compound and an amino compound.
 7. The method of claim6, wherein said amino compound is one of choline, TRIS,bis-tris-propane, N-methylglucamine or arginine.
 8. A method of treatingor preventing the adverse consequences of the hyperactivity of the NMDAreceptor, comprising administering to an animal in need of suchtreatment or prevention an effective amount of a compound having theFormula

or a tautomer or a pharmaceutically acceptable salt thereof; wherein R¹is nitro, cyano, CF₃, carboxy, or alkanoyl; R² is alkoxy, aralkoxy,thioalkyl, hydroxy, mercaptoalkyl, or azido; R³ is halo, haloalkyl,nitro, alkyl, alkoxy, azido, or cyano; and R⁴ is hydrogen.
 9. The methodof claim 8, wherein said compound is selected from the group consistingof 6-azido-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-fluoro-6-methoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-chloromethoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-chloro-6-ethylthio-5-nitro-1,4-dihydroquinoxaline-2,3-dione and7-fluoro-6-ethoxy-5-nitro-1,4-dihydroquinoxaline-2,3-dione.
 10. A methodof treating or preventing the adverse consequences of the hyperactivityof the NMDA receptor, comprising administering to an animal in need ofsuch treatment or prevention an effective amount of a compound havingthe Formula

or a tautomer or a pharmaceutically acceptable salt thereof; wherein R¹is nitro, fluoro, trifluoromethyl or chloro; R² is fluoro, chloro,alkyl, azido, or cyano; R³ is fluoro or chloro; and R⁴ is hydrogen orfluoro; with the proviso that at least one of R¹-R⁴ is fluoro and thatR² is not fluoro when R¹ is nitro.
 11. The method of claim 10, whereinsaid compound is selected from the group consisting of5,6,7,8-tetrafluoro-1,4-dihydroquinoxaline-2,3,-dione,5,6,7-trifluoro-1,4-dihydroquinoxaline-2,3-dione,6-chloro-7-fluoro-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-fluoro-6-bromo-5-nitro-1,4-dihydroquinoxaline-2,3-dione,7-fluoro-6-methyl-5-nitro-1,4-dihydroquinoxaline-2,3-dione,6-chloro-5,7-difluoro-1,4-dihydroquinoxaline-2,3-dione,5-chloro-6,7-difluoro-1,4-dihydroquinoxaline-2,3-dione,5-chloro-6,7,8-trifluoro-1,4 dihydroquinoxaline-2,3-dione and6-chloro-7-fluoro-5-trifluoromethyl-1,4-dihydroquinoxaline-2,3-dione.